Nucleic acids encoding TGF-beta inhibitors and IL-12 and uses thereof

Abstract

The present disclosure provides nucleic acids encoding transforming growth factor inhibitors and IL-12. Further, nucleic acids encoding the chemokine receptor CXCR3 are provided herein. Additionally, oncolytic viruses comprising the nucleic acids described herein are described herein. The compositions described herein are further described for use in the treatment of cancer. Described herein is a composition comprising a vector, the vector comprising an exogenous nucleic acid comprising a sequence encoding a cytokine or a functional variant thereof, an exogenous nucleic acid comprising a sequence encoding a chemokine receptor or a functional variant thereof, and a first promoter region upstream of the sequence encoding the chemokine receptor, causing expression of the chemokine receptor before expression of the cytokine.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application No. 63 / 417,487, filed October 19, 2022, and U.S. Provisional Application No. 63 / 471,811, filed June 8, 2023, both of which are incorporated by reference herein in their entireties.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ST.26 xml format, which is incorporated herein by reference in its entirety. A copy of said xml, created on September 28, 2023, is named 199249-720601_SL.xml and is 110,350 bytes in size. [Background technology]

[0003] background Cancer is a major source of disease, especially in developed countries. Transforming growth factor beta ("TGF-β," "TGF-beta," or "TGF-b") is highly overexpressed by many cancer cell types, providing a means of evading the host immune response. Therefore, there is a need for compositions and methods for targeting and modulating TGF-b activity in the microenvironment. Summary of the Invention [Means for solving the problem]

[0004] overview Described herein is a composition comprising a vector, the vector comprising an exogenous nucleic acid comprising a sequence encoding a cytokine or a functional variant thereof, an exogenous nucleic acid comprising a sequence encoding a chemokine receptor or a functional variant thereof, and a first promoter region upstream of the sequence encoding the chemokine receptor, the first promoter region causing expression of the chemokine receptor before expression of the cytokine.

[0005] Described herein are nucleic acids comprising sequences encoding at least two polypeptides, including interleukin-12 (IL-12) or a functional variant thereof and an inhibitor of transforming growth factor beta (TGF-beta) activity.

[0006] Described herein are nucleic acids comprising a first region encoding a first polypeptide comprising a sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:12 or SEQ ID NO:15, and a second region encoding a second polypeptide comprising a sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:40 or SEQ ID NO:41.

[0007] Described herein are nucleic acids, wherein the nucleic acid molecule comprises an insertion at the A52R locus, the insertion comprising, in 5' to 3' order, a first promoter region whose promoter comprises the A52R promoter, a first region encoding human CXCR3, a second promoter region whose promoter comprises P135, a second region encoding human IL-12, a third promoter region whose promoter comprises P7.5, and a third region encoding a TGF beta variant.

[0008] Described herein are nucleic acids, wherein the nucleic acid molecule comprises an insertion at the A52R locus comprising the sequence set forth in SEQ ID NO:88, an insertion at the TK locus comprising the sequence set forth in SEQ ID NO:85.

[0009]

[0010] Described herein are pharmaceutical compositions comprising a nucleic acid described herein or a vector described herein and a pharmaceutically acceptable excipient.

[0011] Described herein are methods for the treatment of cancer, the methods comprising administering to a subject having cancer a pharmaceutical composition described herein in an amount sufficient to treat the cancer.

[0012] Described herein are methods for activating an anti-tumor immune response, the method comprising administering to a subject with cancer a pharmaceutical composition described herein.

[0013] Described herein is a method for reducing the incidence of tumor cell growth, comprising administering to tumor cells a pharmaceutical composition described herein in an effective amount sufficient to reduce the incidence of tumor cell growth.

[0014] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description, which sets forth illustrative embodiments, and the accompanying drawings, in which the principles of the disclosure are utilized. [Brief explanation of the drawings]

[0015] [Figure 1] Figures 1A and 1B show the interaction of TGF-β receptor subunits I (RI) and II (RII) with native and modified TGF-beta ligands. Figure 1A illustrates that unmodified TGF-β binds to RII and RI to generate a receptor complex. Figure 1B shows that the modified TGF-β described herein binds to RII and has no ability to bind to RI, preventing the formation of the receptor complex.

[0016] [Figure 2] Figure 2 shows a diagram of an expression construct having a P7.5 promoter driving expression of an IL-12 polypeptide comprising the IL-12 beta and alpha subunits linked by a 22-residue glycine-rich linker, and a P28 promoter driving expression of a TGFbf polypeptide comprising the signal peptide of interleukin-2 (IL-2sig) fused to a modified TGF-beta variant in which cysteines 8 and 17 have been mutated to valine and arginine, respectively.

[0017] [Figure 3] Figure 3A is a line graph showing the mean volume of Renca cell tumors induced on the y-axis against days post-treatment on the x-axis in mice after treatment with buffer control, TK-control, or viruses expressing IL-12, TGFbf, or both IL-12 and TGFbf.

[0018] Figure 3B is a line graph showing the mean volume of induced B16 cell tumors on the y-axis versus days post-treatment on the x-axis in mice after treatment with buffer control, TK-control, or viruses expressing murine IL-12, TGFbf, or both IL-12 and TGFbf.

[0019] [Figure 4] Figure 4A is a line graph showing the probability of survival on the y-axis versus time in days on the x-axis in mice with Renca cell tumors induced and treated with PBS (1), TK-control (2), or viruses expressing murine IL-12 (3), both murine IL-12 and TGFbf1 (4), or TGFbf1 (5).

[0020] Figure 4B is a line graph showing the probability of survival on the y-axis versus days on the x-axis in mice with induced B16 cell tumors treated with PBS (1), TK-control (2), or viruses expressing murine IL-12 (3), both murine IL-12 and TGFbf1 (4), or TGFbf1 (5).

[0021] [Figure 5] FIG. 5 shows a diagram of an expression construct with the A52R promoter driving expression of CXCR3.

[0022] [Figure 6] Figure 6 shows a diagram of an expression construct having the 135 promoter driving expression of an IL-12 polypeptide comprising IL-12 beta and alpha subunits linked by a 22-residue glycine-rich linker and the P7.5 promoter driving expression of the TGF-beta variant, TGF-bv2.

[0023] [Figure 7] FIG. 7 shows the insertion of sequences expressing CXCR3 in the A52R locus and sequences expressing IL-12 and TGF-beta inhibitors in the tyrosine kinase locus.

[0024] [Figure 8-1] Figure 8A is a one-dimensional histogram obtained by FACS analysis of HeLa cells infected with vaccinia virus modified to express CXCR3, IL-12, and TGF-beta inhibitor (TGFBi / IL-12 / CXCR3), infected with a control virus (control), or uninfected (-), showing increased detection of expressed CXCR3 in HeLa cells infected with the modified virus.

[0025] [Figure 8-2] Figures 8B-8E are bar plots showing migration of the indicated peripheral blood monocyte (PBMC) populations toward the CXCR3 ligand CXCL11 after infection with vaccinia virus modified to express CXCR3, IL-12, and TGF-beta inhibitor (TGFBi / IL-12 / CXCR3), control virus (control), or no infection (-). Figure 8B shows CD4 cell migration, Figure 8C shows CD8 cell migration, Figure 8D shows monocyte migration, and Figure 8E shows B cell migration.

[0026] Figure 8F is a bar plot showing quantitative ELISA detection of IL-12 in Hela supernatants obtained from cells infected with vaccinia viruses modified to express CXCR3, IL-12, and TGF-beta inhibitors, infected with a control virus, or without infection, showing that expression is only detectable in supernatants obtained from Hela cells infected with the modified viruses.

[0027] Figure 8G is a photograph of a Western blot obtained from Hela lysates infected with vaccinia viruses modified to express CXCR3, IL-12, and TGF-beta inhibitor, or infected with a control virus, or without infection, showing a 12 kDa band corresponding to TGF-beta inhibitor that is detectable only in lysates from cells infected with the modified virus.

[0028] [Figure 9] Figure 9A shows traces of fluorescence incorporation in several generations of cells, represented by peaks, in CD8 T cells infected with vaccinia virus modified to express CXCR3, IL-12, and TGF-beta inhibitor (TGFBi / IL-12 / CXCR3), infected with a control virus (control), or treated with no infection (-), 0 ng / ml, 10 ng / ml, or 50 ng / ml of TGF-beta1, demonstrating that infection with the modified virus inhibits TGF-beta suppression.

[0029] Figure 9B provides a FACS analysis plot of CD8 cells screened for CD44 and granzyme B (GZMB) after infection with vaccinia viruses modified to express CXCR3, IL-12, and TGF-beta inhibitor (TGFBi / IL-12 / CXCR3), TK-control virus (control), or no infection (-), demonstrating increased expression of GZMB in cells infected with the modified viruses.

[0030] [Figure 10] Figure 10A is a bar plot showing the PFU / ml detected in human lung adenocarcinoma (A549) cells, human cervical carcinoma (Hela) cells, and human foreskin fibroblasts (HFF) after infection with vaccinia virus modified to express CXCR3, IL-12, and a TGF-beta inhibitor, showing an increase in virus in HeLa cells.

[0031] Figure 10B is a plot showing the viral genome copies per mg of tumor detected in RENCA tumors after infection with vaccinia viruses modified to express CXCR3, IL-12, and a TGF-beta inhibitor compared to unmodified viruses, demonstrating increased viral counts in tumors infected with the modified viruses.

[0032] [Figure 11] Figures 11A and 11B are plots of tumor size over 51 days in EMT6 (Figure 11A) and MC38 (Figure 11B) tumor models infected with vaccinia virus modified to express CXCR3, IL-12, and TGF-beta inhibitor (TGFBi / IL-12 / CXCR3), with a control virus (control), or without infection (-), showing almost complete inhibition of growth in tumors infected with the modified viruses.

[0033] Figures 11C and 11D are plots of survival probability in EMT6 (Figure 11C) and MC38 (Figure 11D) tumor models infected with vaccinia viruses modified to express CXCR3, IL-12, and TGF-beta inhibitor (TGFBi / IL-12 / CXCR3), TK-control virus, or no infection (-), showing increased survival in tumors infected with the modified viruses.

[0034] [Figure 12-1] Figure 12A shows photographs of prepared RENCA and MC38 tumor samples treated with CD3, CD8, and nuclear staining after infection with vaccinia virus modified to express CXCR3, IL-12, and TGF-beta inhibitor (TGFBi / IL-12 / CXCR3), TK-control virus (control), or no infection (-), demonstrating increased infiltration of CD3 and CD8 cells into the tumor after infection with the modified viruses.

[0035] [Figure 12-2]Figures 12B-12E are bar plots of the total counts of CD3+ and CD8+ T cells in RENCA and MC38 tumor samples after infection with vaccinia viruses modified to express CXCR3, IL-12, and TGF-beta inhibitor (TGFBi / IL-12 / CXCR3), TK-control virus (control), or no infection (-). Figure 12B shows increased CD3+ cells in RENCA tumors infected with the modified viruses. Figure 12C shows increased CD8+ cells in RENCA tumors infected with the modified viruses. Figure 12D shows increased CD3+ cells in MC38 tumors infected with the modified viruses. Figure 12E shows increased CD8+ cells in MC38 tumors infected with the modified viruses.

[0036] [Figure 13] Figure 13A is a heat map showing the relative expression levels of type II interferon gamma (INFG)-related genes compared to the overall average in cells treated with vaccinia viruses modified to express CXCR3, IL-12, and TGF-beta inhibitors and control cells, demonstrating increased expression of INFG-related genes in cells treated with the modified viruses.

[0037] Figure 13B is a heat map showing the relative expression levels of TGF-beta 1-related genes compared to the overall average in cells treated with vaccinia viruses modified to express CXCR3, IL-12, and TGF-beta inhibitors and control cells, demonstrating increased expression of TGF-beta 1-related genes in cells treated with the modified viruses. DETAILED DESCRIPTION OF THE INVENTION

[0038] Detailed Description TGF-beta 1 (TGFB1)-mediated immune resistance is one of the major mechanisms of immunosuppression utilized across multiple tumor types. The immune resistance conferred by TGFB1 can be mediated through its pleiotropic effects on vasculature, fibrosis, and regulatory / effector immune cells within the tumor microenvironment. Blocking TGFB1 with TGF-beta inhibitors (TGFBi) can improve responses to immunotherapy. Furthermore, IL-12 is a cytokine that can promote type 1 inflammatory responses, M1 macrophage skewing, and effector CD8 T cell responses through IFNg induction. Combining TGFB1 blockade with IL-12 can enhance therapeutic efficacy by simultaneously reducing immune suppression and enhancing antitumor immune responses. In addition, CXCR3 expression from the viral backbone can enhance systemic viral delivery to tumors rich in CXCR3 ligands.

[0039] Current TGF-beta inhibitors have challenges in specificity and delivery. For example, receptor-targeted small molecule receptor kinase inhibitors (SMRKIs) tend to have low specificity and selectivity. Antibodies and receptor traps have been reported to penetrate dense tissues, such as tumors, poorly. As a solution to these problems, the TGF-beta variants provided herein offer specific receptor targeting and improved tissue penetration. The small size of the polypeptide allows for increased tissue penetration. Furthermore, as described herein, delivery can be further enhanced by providing a nucleic acid encoding the TGF-beta inhibitor in a delivery vector. In addition, as described herein, delivery of a TGF-beta inhibitor in combination with IL-12 has shown better overall response and survival.

[0040] Provided herein are compositions and methods for vaccinia-based immunotherapy that combine enhanced systemic viral delivery to CXCR3 ligand-rich tumors with localized expression of IL-12 and TGFBi within the tumor microenvironment. In some embodiments, the methods include cancer treatment. The compositions described herein may contain one or more nucleic acids encoding the polypeptides described herein. The nucleic acids provided herein may include DNA, RNA, nucleic acid analogs, or any combination thereof. Briefly, (1) compositions for the expression of TGFβ inhibitors and IL-12, (2) combinations of nucleic acids, (3) compositions for the expression of chemokine receptors, (4) vectors for the expression of modified nucleic acids, (5) modified oncolytic viruses, (6) treatment conditions, and (7) dosages, forms, and methods of administration of the compositions described herein.

[0041] definition The terminology used herein is for the purpose of describing particular cases only and is not intended to be limiting. As used herein, the singular forms "a," "an," and "the" include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent the terms "contains," "containing," "including," "includes," "having," "has," "with," or variations thereof, are used in either the detailed description and / or claims, such terms are intended to be inclusive in the same manner as the term "comprising."

[0042] The term "about" or "approximately" can mean within an acceptable range of error for a particular value, as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. When a particular value is described in this application and claims, unless otherwise indicated, the term "about" should be assumed to mean within an acceptable range of error for that particular value, e.g., ±10% of the value modified by the term "about."

[0043] The terms "heterologous nucleic acid sequence" or "exogenous nucleic acid sequence" or "transgene" as used herein in reference to a particular virus may refer to a nucleic acid sequence that originates from a source other than the designated virus.

[0044] The term "mutation," as used herein, can refer to a deletion, insertion of a heterologous nucleic acid, inversion, or substitution, including open reading frame removal mutations as generally understood in the art.

[0045] The term "gene," as used herein, may refer to a segment of nucleic acid that encodes a particular protein or RNA (also referred to as a "coding sequence" or "coding region"), optionally along with associated regulatory regions, e.g., promoters, operators, terminators, etc., which may be located upstream or downstream of the coding sequence.

[0046] As used herein, a "promoter" may refer to a regulatory sequence, which is a region of a nucleic acid sequence that controls the initiation and rate of transcription. In certain embodiments, a promoter may include genetic elements to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, can bind. The terms "operably positioned," "operably linked," "under control," and "under transcriptional control" may mean that the promoter is in the correct functional position and / or orientation relative to the nucleic acid sequence so as to control the initiation and / or expression of the transcription of the nucleic acid sequence. In certain embodiments, a promoter may or may not be used in conjunction with an "enhancer," which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.

[0047] The term "homology" as used herein can refer to the calculation of "homology" or "percent homology" between two or more nucleotide or amino acid sequences, which can be determined by aligning the sequences for optimal comparison (e.g., gaps may be introduced into the sequence of the first sequence). The nucleic acids at corresponding positions can then be compared, and the percent identity between the two sequences can be a function of the number of identical positions shared by the sequences (i.e., % homology = number of identical positions / total number of positions × 100). For example, a position in the first sequence can be occupied by the same nucleic acid as the corresponding position in the second sequence, in which case the molecules are identical at that position. The percent homology between two sequences can be a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences. In some embodiments, the length of sequences aligned for comparison purposes may be at least about 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 95% of the length of the reference sequence. A BLAST® search can be used to determine homology between two sequences. The homology can be between the entire length of the two sequences or between fractions of the entire length of the two sequences. The two sequences may be genes, nucleic acid sequences, protein sequences, peptide sequences, amino acid sequences, or fragments thereof. The actual comparison of the two sequences can be accomplished by well-known methods, for example, using a mathematical algorithm. When using BLAST and Gapped BLAST programs, any relevant parameters of the respective programs (e.g., NBLAST) can be used. For example, parameters for sequence comparison can be set at score=100, word length=12, or may be varied (e.g., W=5 or W=20). Other examples include the algorithm of Myers and Miller, CABIOS (1989), ADVANCE, ADAM, BLAT, and FASTA.

[0048] The term "subject" may refer to an animal, including, but not limited to, a primate (e.g., a human), cow, sheep, goat, horse, dog, cat, rabbit, rat, or mouse. The terms "subject" and "patient" are used interchangeably herein, e.g., in reference to a mammalian subject, e.g., a human subject.

[0049] The terms "treat," "treating," and "treatment" may be intended to include alleviating or arresting a disorder, disease, or condition, or one or more of the symptoms associated with a disorder, disease, or condition, or alleviating or eradicating the cause of the disorder, disease, or condition itself.

[0050] The term "therapeutically effective amount" may refer to that amount of a compound that, when administered, may be sufficient to prevent the occurrence of, or alleviate to some extent, one or more of the symptoms of the disorder, disease, or condition being treated.

[0051] The term "oncolytic" as used herein can refer to the killing of cancer or tumor cells by an agent, such as an oncolytic poxvirus, for example, an oncolytic vaccinia virus, through the direct lysis of cancer or tumor cells, for example, by stimulating an immune response against cancer or tumor cells, apoptosis, the expression of toxic proteins, the cessation of autophagy and protein synthesis, the induction of anti-tumor immunity, or any combination thereof. The direct lysis of cancer or tumor cells infected with an agent, such as an oncolytic vaccinia virus, can be the result of viral replication within the cells. In certain instances, the term "oncolytic" can refer to the killing of cancer or tumor cells without the lysis of the cells.

[0052] The term "oncolytic virus" as used herein may refer to a virus that preferentially infects and kills tumor cells. In some embodiments, oncolytic viruses can include, but are not limited to, (i) viruses that naturally replicate preferentially in cancer cells and are non-pathogenic in humans, often due to their high susceptibility to natural antiviral signaling or their dependency on tumorigenic signaling pathways, and (ii) viruses that have been genetically engineered for use. In some embodiments, oncolytic viruses can be measles viruses, polioviruses, poxviruses, vaccinia viruses, adenoviruses, adeno-associated viruses, herpes simplex viruses, vesicular stomatitis viruses, reoviruses, Newcastle disease viruses, Seneca viruses, lentiviruses, mengoviruses, or myxoma viruses. In certain embodiments, oncolytic viruses can be poxviruses. In certain embodiments, oncolytic viruses can be vaccinia viruses.

[0053] The term "modified oncolytic virus," as used herein, may refer to an oncolytic virus that includes modifications to its components, such as, but not limited to, modifications in the virus's native genome ("backbone"), such as mutations or deletions of viral genes, introduction of exogenous nucleic acid, chemical modification of viral nucleic acid or viral proteins, and introduction of exogenous or modified viral proteins into the viral capsid. Generally, oncolytic viruses may be modified (also known as "engineered") to achieve improved therapeutic effects on tumor cells. In some embodiments, the modified oncolytic virus may be a modified poxvirus. In some embodiments, the modified oncolytic virus may be a modified poxvirus. In some embodiments, the modified oncolytic virus may be a modified vaccinia virus.

[0054] The terms "systemic delivery" and "systemic administration," used interchangeably herein, may refer, in some cases, to the route of administration of a pharmaceutical, oncolytic virus, or other substance into the circulatory system. Systemic administration may include oral administration, intraperitoneal administration, parenteral administration, intranasal administration, sublingual administration, rectal administration, transdermal administration, intra-arterial administration, or any combination thereof.

[0055] TGF-beta activity inhibitor The cytokine TGF-beta, in its isoforms TGF-beta1, TGF-beta2, and TGF-beta3, is a potent suppressor of immunity. TGF-beta has been reported to suppress the proliferation of cytotoxic T-lymphocytes (CTLs), natural killer (NK) cells, and dendritic cells (DCs). The cytokine also stimulates the proliferation and activation of regulatory T cells (Tregs).

[0056] Provided herein is a TGF-beta activity inhibitor for the treatment of cancer and related symptoms.TGF-beta signaling activity affects the progression of many diseases, including cancer.TGF-beta binds to TGF-beta RII, and then recruits TGF-beta RI, thereby assembles the receptor TGF-beta RI and TGF-beta RII into a signal transduction tetramer.Preventing the binding to the receptor or its assembly blocks the TGF-beta signal transduction cascade.Provided herein is an inhibitor that prevents the recruitment of TGF-beta RI.

[0057] Compositions comprising nucleic acids encoding TGF-beta activity inhibitors are provided herein. In some embodiments, the TGF-beta inhibitors can bind to TGF-beta receptor II and antagonize TGF-beta expression (FIG. 1B). Without being bound by theory, in some embodiments, the TGF-beta inhibitors specifically target and antagonize their cognate receptors. In further embodiments, the TGF-beta inhibitors have fewer adverse effects associated with off-target activity. Nucleic acids expressing the TGF-beta inhibitors described herein can provide increased tissue penetration. In some embodiments described herein, the TGF-beta inhibitors bind to TGFb receptor II. In some embodiments, the TGF-beta inhibitors do not bind to TGFb receptor I.

[0058] Native TGF-beta is a dimer of two identical 112-residue peptides linked by a disulfide bond. In some embodiments, the nucleic acids described herein encode human TGF-beta monomer, or any of the three isoforms TGF-beta 1, TGF-beta 2, or TGF-beta 3, as set forth in Table 1 by SEQ ID NOS: 1-3, or functional variants thereof. In some embodiments, the nucleic acids described herein encode engineered minimonomers of TGF-beta 1, TGF-beta 2, and TGF-beta 3, or functional variants thereof, as set forth by SEQ ID NOS: 4, 5, and 9. In some embodiments, the engineered minimonomers of TGF-beta 2 described herein comprise a deletion of the alpha 3 helix at residues 52-71 and a substitution of Cys-77 with serine, with amino acid numbering based on SEQ ID NOS: 2. In some embodiments, the engineered minimonomers of TGF-beta 2 comprise a modification to include the cystine-knot region of Dan and Cerberus-related protein (PRDC). In some embodiments, the engineered minimonomer of TGF-beta 2 lacks the alpha-3 heel helix of native TGF-beta. Exemplary sequences for inclusion in the compositions described herein are listed in Table 1, SEQ ID NOS: 4-9.

[0059] Exemplary variants of TGF-beta 2 minimonomer are described by SEQ ID NOs: 6, 7, and 8. Additionally, provided herein are nucleic acids encoding minimonomer variants that contain substitutions that allow for increased solubility and high binding affinity. Table 1. Sequences of TGF-beta isoforms, monomers, and minimonomers [Table 1]

[0060] In some embodiments, the nucleic acids described herein encode TGF-beta inhibitors, including antibodies or functional fragments thereof. In some embodiments, the nucleic acids described herein encode antibodies or functional fragments thereof that bind to TGF-beta. Non-limiting examples of antibodies that bind to TGF-beta include 2G7, 1D11, GC1008, LY2382770, and TbetaM1. In some embodiments, the nucleic acids described herein encode antibodies or functional fragments thereof that bind to TGF-beta receptors. In some embodiments, the antibodies or functional fragments thereof bind to TGF-beta receptor I, TGF-beta receptor II, or TGF-beta receptor III.

[0061] In some embodiments, the nucleic acid described herein encodes a TGF-beta inhibitor comprising a receptor trap. In some embodiments, the encoded receptor trap blocks the entire receptor binding interface. In some embodiments, the encoded receptor trap is an affinity-optimized, soluble variant of the extracellular binding domain. In some embodiments, the target ligand preferentially binds to the receptor trap. In some embodiments, the encoded receptor trap prevents the ligand from binding to the receptor. In some embodiments, the encoded TGF-beta inhibitor regulates signal transduction with the receptor.

[0062] In some embodiments, the nucleic acids described herein encode a TGF-beta inhibitor comprising a peptide. The peptide, in non-exclusive embodiments, comprises P144 or P17. In selected embodiments, the nucleic acids described herein encode a TGF-beta inhibitor comprising a sequence set forth in Table 2. In some embodiments, the nucleic acids described herein encode a TGF-beta inhibitor peptide set forth by SEQ ID NO: 10 or 11. In some embodiments, the nucleic acids described herein encode a TGF-beta inhibitor that binds to TGF-beta. In further embodiments, the TGF-beta-TGF-beta inhibitor complex does not bind to a TGF-beta receptor. The peptide optionally binds to an isoform of TGF-beta. Table 2. Inhibitory peptide sequences. [Table 2]

[0063] In some embodiments, the nucleic acids described herein encode TGF-beta inhibitors comprising dominant-negative receptors. In some embodiments, the dominant-negative receptors comprise truncated TGF-beta receptor I, receptor II, or receptor III. In some embodiments, the nucleic acids described herein encode dominant-negative receptors comprising truncated TGF-beta receptor II. In some embodiments, the dominant-negative receptors are soluble. In some embodiments, the encoded dominant-negative receptors are versions of TGF-beta receptor I, receptor II, or receptor III that lack the transmembrane region.

[0064] In some embodiments, the nucleic acids described herein encode TGF-beta inhibitors, including dominant-negative inhibitors. In some embodiments, the encoded dominant-negative inhibitors bind to TGF-beta receptor I, receptor II, or receptor III. In some embodiments, the encoded dominant-negative inhibitors bind to TGF-beta receptor II. In some embodiments, the encoded dominant-negative inhibitors interfere with TGF-beta inhibitors, optionally including variants of TGF-beta 1, TGF-beta 2, or TGF-beta 3. Non-exclusive embodiments of the nucleic acids encoded herein bind to the ligand-binding domain of TGF-beta receptor II.

[0065] Provided herein is a composition comprising a nucleic acid encoding TGF-beta 2 variant (TGFbv1).In some embodiments, the nucleic acid sequence encodes a peptide described by SEQ ID NO:7.In some embodiments, the encoded TGFbv1 comprises at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity with SEQ ID NO:7.

[0066] Provided herein is a composition comprising a nucleic acid encoding a TGF-beta 2 variant (TGFbv2). In some embodiments, the nucleic acid sequence encodes a peptide described by SEQ ID NO: 8. In some embodiments, the encoded TGFbv2 comprises at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to SEQ ID NO: 8.

[0067] TGF-beta can suppress the proliferation of stimulated immune cells, such as T cells, below baseline levels. Compositions comprising the TGF-beta inhibitors described herein can counteract the suppression caused by TGF-beta and allow stimulated immune cells to proliferate at levels close to baseline. In some embodiments, contacting cells suppressed by TGF-beta with the TGF-beta inhibitors described herein can result in proliferation at about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% of the baseline in unsuppressed cells.

[0068] TGF-beta inhibitors can modulate the expression of a family of related genes. In some embodiments, modulation includes inducing gene expression. In some embodiments, contacting cells with a TGF-beta inhibitor described herein induces expression in one or more interferon gamma (IFNG)-related genes compared to the median. In some embodiments, the expression of one or more IFNG-related genes is increased by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 120%, about 140%, about 160%, about 180%, about 200%, about 250%, about 300%, about 350%, or about 400% compared to the median expression across treated and untreated cells. The IFNG-related gene is selected from the group consisting of CXCL11, XCR1, STAT1, IDO1, IL12B, IFNG, CIITA, H2-EB1, H2-AB1, TBX21, CXCR3, CD2, LTB, CXCL16, B2M, VCAM1, TAP1, IFIT2, TAP2, IL2RG, STAT2, CD274, and IRF1.

[0069] In some embodiments, contacting cells with a TGF-beta inhibitor described herein reduces the expression of TGF-B1-related genes compared to the median level. In some embodiments, the expression of TGF-B1-related genes is reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to the median level of expression across treated and untreated cells. The TGF-B1-related gene is selected from the group consisting of ILz1B, LPL, SLP1, FBN1, LCN2, CXCL5, OGN, PLOD2, TNFAIP6, CAN, ABCG1, ACKR3, and COL15A1.

[0070] Granzyme B

[0071] Granzyme B (GZMB) is found in the granules of immune cells, such as natural killer cells (NK cells) and cytotoxic T cells. It is secreted by these cells and can mediate apoptosis in target cells. GZMB can also be produced by non-cytotoxic cells, such as basophils and mast cells. This can help induce inflammation and extracellular matrix degradation. Activated immune cells can exhibit increased GZMB expression. Compositions containing the TGF-beta inhibitors described herein can prevent suppression by TGF-beta and allow stimulated immune cells to express GZMB.

[0072] Contacting immune cells with the modified viruses described herein can induce expression of granzyme B (GZMB) greater than that in untreated cells. In some embodiments, cells contacted with a TGF-beta inhibitor contain about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 125%, about 150%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000% more GZMB than untreated cells.

[0073] IL-12 Compositions comprising nucleic acids encoding IL-12 or functional variants thereof are provided herein. In some embodiments, the nucleic acids described herein encode at least two polypeptides. In some embodiments, the nucleic acids encode a first polypeptide comprising interleukin-12 or a functional variant thereof. In some embodiments, the nucleic acids encode IL-12. In further embodiments, the IL-12 comprises subunit beta (IL-12b) and subunit alpha (IL-12a). In some embodiments, the nucleic acid encodes the mouse IL-12 (mIL-12) sequence described by SEQ ID NO: 12. In some cases, the encoded mIL-12 comprises at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to SEQ ID NO: 12. In some embodiments, the nucleic acid encodes a human IL-12 (hIL-12) sequence set forth by SEQ ID NO: 15. In some cases, the encoded hIL-12 comprises at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to SEQ ID NO:15.

[0074] Provided herein are compositions comprising a nucleic acid encoding mouse IL-12 subunit alpha (IL-12a) (UniProtKB Accession ID 43431.1). In some embodiments, the nucleic acid sequence encodes a peptide described by SEQ ID NO: 13. In some cases, the encoded IL-12a comprises at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to SEQ ID NO: 13.

[0075] Provided herein are compositions comprising a nucleic acid encoding mouse IL-12 subunit beta (IL-12b) (UniProtKB Accession ID P43432.1). In some embodiments, the nucleic acid sequence encodes a peptide described by SEQ ID NO: 14. In some cases, the encoded IL-12b comprises at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to SEQ ID NO: 14.

[0076] Provided herein are compositions comprising a nucleic acid encoding human IL-12 subunit alpha (hIL-12a) (UniProtKB Accession ID P060595). In some embodiments, the nucleic acid sequence encodes a peptide described by SEQ ID NO: 16. In some cases, the encoded hIL-12a comprises at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to SEQ ID NO: 16.

[0077] Provided herein are compositions comprising a nucleic acid encoding human IL-12 subunit beta (hIL-12b) (UniProtKB Accession ID P29460). In some embodiments, the nucleic acid sequence encodes a peptide described by SEQ ID NO: 17. In some cases, the encoded IL-12b comprises at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to SEQ ID NO: 17.

[0078] Exemplary amino acid sequences of regions of IL-12 for inclusion in the compositions described herein are listed in Table 3. Table 3. IL-12 amino acid sequence. [Table 3-1] [Table 3-2]

[0079] Linker and signaling domains Compositions comprising nucleic acids encoding linkers are provided herein. In some embodiments, the nucleic acid encoding the linker is disposed between various encoded biologically functional units described herein. In some embodiments, the encoded linker is flexible or rigid. In further embodiments, the encoded linker is a cleavable linker. In further embodiments, the encoded cleavable linker comprises a disulfide bond. In further embodiments, the encoded cleavable linker comprises a protease-sensitive domain. A non-limiting list of exemplary linkers encoded by nucleic acids included in the compositions described herein is listed in Table 3. In some embodiments, the compositions described herein comprise a nucleic acid encoding a linker having a sequence set forth by SEQ ID NO: 18. In some embodiments, the nucleic acid encodes a linker comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 18. Table 4. Linkers. [Table 4-1] [Table 4-2] Subscripts in the sequences indicate repeats. Arrows in the sequence indicate the location of the cleavage site.

[0080] The present invention provides a composition comprising a nucleic acid encoding a signal transduction domain for controlling cell function.The activity of interleukins, such as, but not limited to, IL-2 and IL-15, depends on the processing of signal peptides.The present invention provides a composition comprising a nucleic acid encoding at least one signal peptide.Table 5 shows non-limiting examples of signal peptides encoded by nucleic acids in the compositions described herein. Table 5. Signal sequences. [Table 5]

[0081] Provided herein are compositions comprising a nucleic acid encoding the mouse IL-2 signal sequence (mIL-2sig) (UniProtKB Accession ID P04351.1). In some embodiments, the nucleic acid sequence encodes mIL-2sig corresponding to SEQ ID NO: 36, or a functional variant thereof. In some embodiments, the encoded mIL-2sig comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 36.

[0082] Provided herein are compositions comprising a nucleic acid encoding a human IgE signal sequence (hIgEsig). In some embodiments, the nucleic acid sequence encodes a hIgEsig corresponding to SEQ ID NO: 37 or a functional variant thereof. In some embodiments, the encoded hIgEsig comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 37.

[0083] Combined Nucleic Acid Sequences Compositions comprising nucleic acids encoding a combination of technical properties are provided herein. In some embodiments, the nucleic acid encodes IL-12 or a functional variant thereof, a signal peptide, and a TGF-beta variant polypeptide. In some embodiments, the nucleic acid encodes IL-12, a mouse IL-2 signal peptide, and a TGF-beta variant polypeptide or a functional variant thereof. In some embodiments, the nucleic acid encodes IL-12, a human IgE signal peptide, and a TGF-beta variant polypeptide or a functional variant thereof.

[0084] In alternative embodiments, two nucleic acids are provided, wherein a first nucleic acid encodes a first polypeptide comprising interleukin-12 or a functional variant thereof, and a second nucleic acid encodes a second polypeptide comprising a TGF-beta fusion (TGFbf) polypeptide comprising a signal peptide and a TGF-beta variant. In some embodiments, the TGFbf polypeptide comprises the sequences set forth in SEQ ID NO:36 and SEQ ID NO:7 (TGFbf1). In some embodiments, the TGFbf polypeptide comprises the sequences set forth in SEQ ID NO:37 and SEQ ID NO:8 (TGFbf2).

[0085] Compositions comprising a nucleic acid sequence encoding a polypeptide are provided herein. In some embodiments, the nucleic acid encodes a polypeptide comprising TGFbf1, as set forth in SEQ ID NO: 40 and shown in Table 6. In some embodiments, the encoded TGFbf comprises at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to SEQ ID NO: 40. In some embodiments, the nucleic acid encodes a polypeptide comprising TGFbf2, as set forth in SEQ ID NO: 41 and shown in Table 6. In some embodiments, the encoded TGFbf comprises at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to SEQ ID NO:41. Table 6. TGF-beta fusion amino acid sequences. [Table 6] chemokine receptors

[0086] Chemokines are chemotactic cytokines that regulate cell trafficking and positioning by activating seven-transmembrane chemokine receptors. Chemokines are sometimes divided into four subfamilies, including the CC, CXC, CX3C, and XC subfamilies, based on the location of their first two N-terminal cysteine ​​residues. Differential expression of chemokine receptors on leukocytes results in the selective recruitment of specific cell types under specific conditions, as needed, to provide an appropriate and efficient immune response to infectious agents or foreign damage. In addition to their pivotal role in the coordinated migration of immune cells to sites of inflammation, chemokines often also play important roles in lymphoid tissue development, immune cell maturation, and the generation and delivery of adaptive immune responses.

[0087] Tumors are increasingly recognized as complex microenvironments formed by numerous different cell types that coexist and communicate with each other in complex signaling networks. Chemokines are essential regulators of cell migration and cell-cell interactions and therefore have a profound impact on tumorigenesis. In the tumor microenvironment, tumor-associated host cells and cancer cells release a range of different chemokines, leading to the recruitment and activation of different cell types that mediate the balance between anti-tumor and pro-tumor responses. In addition to their primary role as chemoattractants, chemokines are often involved in other tumor-related processes, including tumor cell growth, angiogenesis, and metastasis.

[0088] Tumor cells have been shown to acquire the ability to produce growth-promoting chemokines. For example, melanomas have been found to express a number of chemokines, including CXCL1, CXCL2, CXCL3, CXCL8, CCL2, and CCL5, which have been implicated in tumor growth and progression. CCL2 levels can be found elevated in neuroblastoma cell lines and primary tumor cells isolated from human patients. Immunostaining studies have also suggested elevated expression levels of CXCL12 in various cancers, including breast cancer, carcinoid, cervical cancer, colorectal cancer, endometrial cancer, liver cancer, lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, and gastric cancer.

[0089] Chemokine receptors are cytokine receptors found on the surface of certain cells that interact with chemokines. In humans, 20 different chemokine receptors have been discovered. Each has a seven-transmembrane structure and couples to a G protein for intracellular signaling, making it a member of the large protein family of G protein-coupled receptors. After interacting with their specific chemokine ligands, chemokine receptors transduce intracellular calcium (Ca 2+ ) ions (calcium signaling), which triggers a cellular response, including the occurrence of a process known as chemotaxis, which transports cells to a desired location within an organism. Generally, the term "chemokine receptor," as used herein, can refer to a membrane-bound protein that selectively binds to and induces chemotaxis toward a chemokine ligand.

[0090] It should be understood that the chemokine receptors disclosed herein, in some cases, refer not only to naturally occurring chemokine receptors identified in the human body, but also include chemokine receptors derived from other sources, such as, but not limited to, (1) naturally occurring chemokine receptors identified in animals, e.g., pigs, dogs, cattle, and sheep, and (2) non-naturally occurring chemokine receptors, such as mutant proteins, chimeric receptors, and designed proteins having binding affinity for a particular type of chemokine. In some examples, a fragment of a naturally occurring chemokine receptor is also considered a chemokine receptor if the fragment retains the function of binding to and responding to the corresponding chemokine and inducing cellular chemotaxis. As provided herein, in some embodiments, a virus containing an exogenous nucleic acid encoding a chemokine receptor forces virus-infected cells to express the chemokine receptor because the virus hijacks the host cell's gene expression machinery.

[0091] In some embodiments, the present invention provides a modified oncolytic virus comprising an exogenous nucleic acid encoding a chemokine receptor, also referred to herein as a transgene. In some cases, the exogenous nucleic acid is a therapeutic transgene. In some cases, the modified oncolytic virus comprises an exogenous nucleic acid encoding a cytokine receptor whose cognate cytokine is expressed in the tumor microenvironment (e.g., IL15-R has the cognate cytokine IL15 expressed in the tumor microenvironment). In some cases, the modified oncolytic virus encodes a chemokine receptor whose cognate chemokine is likely to be expressed on tumors (e.g., CXCR4 has the cognate chemokine CXCL12 expressed on tumors, and CCR2 has the target CCL2 expressed on tumors), and is delivered systemically as a naked virus. Through systemic delivery, the modified oncolytic virus enters the bloodstream, where it infects lymphocytes, such as B cells, redirecting the infected B cells to tumors and resulting in a significantly increased viral load in tumors. In certain embodiments, increased viral load in tumors is achieved immediately after systemic delivery. The ability to deliver the modified oncolytic viruses disclosed herein in a systemic manner provides advantages over traditional intratumoral delivery methods of oncolytic viruses. Although intratumoral delivery is useful for treating easily accessible tumors, in some cases, it is crucial to treat inaccessible or metastatic cancers, which are considered to be the main cause of death from disease. In this situation, relying on intratumoral delivery of oncolytic viruses is ineffective because it requires systemic dissemination after administration to distant sites. However, this dissemination is often transient and ineffective, at least in part due to the development of an immune response to viral infection.

[0092] Chemokine receptors are divided into different families. Non-limiting examples of chemokine receptors described herein include CXC chemokine receptors, CC chemokine receptors, CX3C chemokine receptors, and XC chemokine receptors, which correspond to four different subfamilies of chemokines that they bind to. Among CXC chemokine receptors, CXCR1 and CXCR2 are closely related, while CXCR1 binds to CXCL8 and CXCL6, CXCR2 binds to CXCL1 and CXCL7, CXCR3 binds to CXCL9, CXCL10, and CXCL11, CXCR4 binds to CXCL12 (or SDF-1), CXCR5 binds to CXCL13, and CXCR6 binds to CXCL16. Among the CC chemokine receptors, ligands for CCR1 include CCL4, CCL5, CCL6, CCL14, CCL15, CCL16, and CCL23; ligands for CCR2 include CCL2, CCL8, and CCL16; ligands for CCR3 include CCL11, CCL26, CCL7, CCL13, CCL15, CCL24, CCL5, CCL28, and CCL18; ligands for CCR4 include CCL3, CCL5, CCL17, and CCL22; and ligands for CCR5 include CCL4, CCL5, CCL17, and CCL22. Ligands for CCR6 include CCL20, CCR7 include CCL19 and CCL21, CCR8 include CCL1 and CCL16, CCR9 include CCL25, CCR10 include CCL27 and CCL28, and CCR11 include CCL19, CCL21, and CCL25. The CX3C chemokine receptor CX3CR1 has the ligand CXCL1. The XC chemokine receptor XCR1 binds to both XCL1 and XCL2.

[0093] Non-limiting embodiments of the present disclosure provide a modified oncolytic virus comprising an exogenous nucleic acid encoding a chemokine receptor. In some embodiments, the chemokine receptor is a CXC chemokine receptor, a CC chemokine receptor, a CX3C chemokine receptor, an XC chemokine receptor, or any combination thereof. In some embodiments, the chemokine receptor is CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, CX3CR1, XCR1, or any combination thereof.

[0094] In certain embodiments, the modified oncolytic virus comprises an exogenous CXCR4-expressing nucleic acid. In certain embodiments, the modified oncolytic virus comprises an exogenous CCR2-expressing nucleic acid. Certain embodiments disclose modified oncolytic viruses comprising exogenous nucleic acids encoding both CXCR4 and CCR2, wherein both chemokines are expressed from the same virus. Under certain circumstances, CXCL12 and / or CCL2, which are typically expressed in the tumor microenvironment, attract lymphocytes expressing CXCR4 and / or CCR2 or other migratory cells infected with the modified oncolytic virus, thereby enhancing tumor-targeted delivery of the modified oncolytic virus. The nucleic acid and amino acid sequences of selected chemokine receptors are listed in Table 7. Table 7. Chemokine receptor sequences. [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4]

[0095] In the compositions provided herein, the oncolytic virus gene may be mutated or replaced with a nucleic acid encoding a chemokine receptor listed in Table 7. In some embodiments, the chemokine receptor is mouse CXCR3 as set forth in SEQ ID NO: 42. In some embodiments, the encoded mouse CXCR3 comprises at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to SEQ ID NO: 42. In some embodiments, the chemokine receptor is human CXCR3 as set forth in SEQ ID NO: 43. In some embodiments, the encoded human CXCR3 comprises at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to SEQ ID NO:43.

[0096] In some embodiments, provided herein is a modified oncolytic virus comprising an exogenous nucleic acid encoding a membrane-associated protein that degrades hyaluronan, such as hyaluronidase. In some embodiments, provided herein is a modified oncolytic virus comprising an exogenous nucleic acid encoding a chemokine receptor, also referred to herein as a transgene. In some cases, the exogenous nucleic acid is a therapeutic transgene.

[0097] promoter Provided herein are compositions comprising nucleic acids, wherein the nucleic acids encode at least one promoter region. A promoter region, or promoter, or promoter element, or regulatory region, refers to a nucleic acid sequence to which a protein binds to initiate transcription. Promoters are typically located 5', or upstream, of the DNA coding region they control. In some embodiments, the nucleic acids described herein comprise one promoter. In some embodiments, one promoter drives the transcription of all polypeptides encoded on the nucleic acid. In some embodiments, the nucleic acids described herein comprise separate promoters for each polypeptide encoded on the nucleic acid. In some embodiments, the nucleic acid comprises two promoters, each of which drives the transcription of one of the two polypeptides encoded on the nucleic acid.

[0098] The timing of expression can be modulated by the promoter structure that regulates gene expression. The number and affinity of transcription factor binding sites determine the relative timing of expression between different promoter regions. Promoters with more transcription factor binding sites and / or higher binding affinity can initiate expression earlier than promoters with fewer or lower affinity binding sites.

[0099] The application of relative temporal expression of proteins can be utilized to express specific factors from the modified viruses described herein either early or late in the infection process. Early promoters have repetitive transcription factor binding sites. Late promoters have fewer binding sites than early promoters. In some embodiments, receptors are expressed using early promoters. Expression early in infection allows for expression and processing by the cell before cellular processes are disrupted. In some embodiments, one or more cytokines are expressed using late promoters.

[0100] In some embodiments, provided herein are promoters including P7.5, P28, P135, TK promoter, A52R promoter, 454 promoter, PB8, LEO, PF11, F7L, H5R, mH5, H1L, A1L, J3R, E4L, I1L, I3L, I4L, I5L, I7L, T7, I2L, FP4b, ATI, P11, PFL1, L4R, T7 promoter, 28kDa promoter, short synthetic promoter (SSP), or any functional variant or combination thereof. In some embodiments, the promoter comprises an early promoter. In some embodiments, the early promoter comprises A52R, PB8, mH5, I4L, LEO, PF11, I3L, P7.5, TK promoter, F7L, H5R, short synthetic promoter (SSP), or any variation or combination thereof. In some embodiments, the promoter comprises a late promoter. In some embodiments, the late promoter comprises SSP, P7.5, P28, P135, TK promoter, F7L, H5R, H1L, A1L, J3R, E4L, I1L, I5L, I7L, T7, I2L, FP4b, ATI, P11, PFL1, L4R, 28kDa promoter, or any functional variant or combination thereof. The sequences of selected promoters are listed in Table 8. Table 8. Promoter nucleic acid sequences [Table 8]

[0101] The compositions provided herein may include a P7.5 promoter and a P28 promoter. In some embodiments, the P7.5 promoter drives expression of a region encoding an IL-12 polypeptide. In some embodiments, the P28 promoter drives transcription of a TGFbf polypeptide. A schematic diagram of the promoter and gene expression construct is shown in FIG. 2. In some embodiments, the P7.5 promoter comprises the nucleic acid sequence set forth in SEQ ID NO: 57. In some embodiments, the P28 promoter comprises the nucleic acid sequence set forth in SEQ ID NO: 58.

[0102] The compositions provided herein may comprise a P135 promoter and a P7.5 promoter. In some embodiments, the P135 promoter drives expression of a region encoding an IL-12 polypeptide. In some embodiments, the P7.5 promoter drives expression of a region encoding a TGF-beta polypeptide. In some embodiments, the P7.5 promoter comprises the nucleic acid sequence set forth in SEQ ID NO: 57. In some embodiments, the P135 promoter comprises the sequence set forth in SEQ ID NO: 56.

[0103] The compositions provided herein may comprise a P7.5 promoter (SEQ ID NO: 57), a P135 promoter (SEQ ID NO: 56), and an A52R promoter. In some embodiments, the P135 promoter drives expression of a region encoding an IL-12 polypeptide. In some embodiments, the P7.5 promoter drives expression of a region encoding a TGF-beta polypeptide. In some embodiments, the A52R promoter drives expression of a region encoding a CXCR3 receptor. In some embodiments, expression of the CXCR3 receptor occurs before expression of an IL-12 polypeptide or a TGF-beta polypeptide.

[0104] Provided herein are compositions comprising an expression construct comprising, in 5' to 3' order, an IL-12 beta subunit, a linker, an IL-12 alpha subunit, an IL-12 signal sequence, and a TGF-beta variant. In some embodiments, the expression construct comprises, in 5' to 3' order, hIL-12b (SEQ ID NO: 17), a flexible linker (SEQ ID NO: 18), hIL-12a (SEQ ID NO: 16), a human IgE sig sequence (SEQ ID NO: 37), and a TGF-beta 2 variant sequence (TGFbv2) (SEQ ID NO: 8). In some embodiments, the expression construct comprises, in 5' to 3' order, mIL-12b (SEQ ID NO: 14), a flexible linker (SEQ ID NO: 18), mIL-12a (SEQ ID NO: 16), a mouse IL-2 sig sequence (SEQ ID NO: 36), and a TGF-beta 2 variant sequence (TGFbv1) (SEQ ID NO: 7). In some embodiments, the expression construct comprises, in 5' to 3' order, mIL-12 (SEQ ID NO: 12) and TGFbf1 (SEQ ID NO: 40). In some embodiments, the expression construct comprises, in 5' to 3' order, hIL-12 (SEQ ID NO: 15) and TGFbf2 (SEQ ID NO: 41). In some embodiments, the exogenous nucleic acids described herein are integrated into a viral genome.

[0105] Provided herein are compositions comprising an expression construct comprising, in 5' to 3' order, an IL-12 signal sequence, a TGF-beta variant, an IL-12 beta subunit, a linker, and an IL-12 alpha subunit. In some embodiments, the expression construct comprises, in 5' to 3' order, a human IgE sig sequence (SEQ ID NO: 37), a TGF-beta 2 variant sequence (TGFbv2) (SEQ ID NO: 8), hIL-12b (SEQ ID NO: 17), a flexible linker (SEQ ID NO: 18), and hIL-12a (SEQ ID NO: 16). In some embodiments, the expression construct comprises, in 5' to 3' order, a mouse IL-2 sig (SEQ ID NO: 36), a TGF-beta 2 variant sequence (TGFbv1) (SEQ ID NO: 7), mIL-12b (SEQ ID NO: 14), a flexible linker (SEQ ID NO: 18), and mIL-12a (SEQ ID NO: 16). In some embodiments, the expression construct comprises, in 5' to 3' order, TGFbf1 (SEQ ID NO: 40) and mIL-12 (SEQ ID NO: 12). In some embodiments, the expression construct comprises, in 5' to 3' order, TGFbf2 (SEQ ID NO: 41) and hIL-12 (SEQ ID NO: 15).

[0106] vector A vector can be used to deliver exogenous nucleic acid to cells for replication or expression. In some embodiments, the vector is a particle containing a plasmid, a viral vector, a cosmid, or an artificial chromosome. A vector generally carries an exogenous nucleic acid inserted into the "backbone" nucleic acid of the vector. In some embodiments, the exogenous nucleic acid is DNA or RNA. In some embodiments, the vector contains at least one promoter sequence that drives the expression of the exogenous nucleic acid.

[0107] Oncolytic viruses Oncolytic viruses can preferentially infect and kill cancer cells. Oncolysis of infected cancer cells can perpetuate the spread of the virus to surrounding tissues and stimulate host anti-tumor immune responses. Provided herein is a composition comprising an oncolytic virus, wherein the oncolytic virus comprises a modified nucleic acid described herein. As used herein, an oncolytic virus kills cancer or tumor cells through mechanisms such as direct lysis of the cells, stimulation of an immune response against the cells, apoptosis, expression of toxic proteins, shutting down autophagy and protein synthesis, induction of anti-tumor immunity, or any combination thereof. In some embodiments, the oncolytic viruses described herein replicate intracellularly. In some embodiments, the oncolytic viruses described herein replicate in tumor cells, immune cells, somatic cells, hematopoietic cells, or another type of cell. Exemplary oncolytic viruses for inclusion in the compositions described herein include, but are not limited to, poxvirus, vaccinia virus, adeno-associated virus, adenovirus, reovirus, lentivirus, herpes simplex virus, vesicular stomatitis virus, mengovirus, myxoma virus, Newcastle disease virus, measles virus, or poliovirus.These oncolytic viruses tend to specifically target cancer cells, and during viral replication, cause significant cell death and tumor regression.In some embodiments, the oncolytic virus is vaccinia virus. Exemplary vaccinia viruses include, without limitation, the following strains, with respect to modification by inclusion of the constructs described herein: Western Reserve vaccinia virus (ATCC VR-1354), vaccinia virus Ankara (ATCC VR-1508), vaccinia virus Ankara (ATCC VR-1566), vaccinia virus strain Wyeth (ATCC VR-1536), or vaccinia virus Wyeth (ATCC VR-325). Further, in some embodiments, the recombinant vaccinia virus is a modified version of a wild-type or attenuated vaccinia virus strain.Non-limiting examples of vaccinia virus strains include the Western Reserve, Copenhagen, IHD, Wyeth (NYCBOH), Tian Tan, Lister, USSR, Ankara, NYVAC, Ankara (MVA), Paris, Bern, Temple of Heaven, Dairen, EM-63, Evans, King, Patwadangar, or Tash Kent strains of vaccinia virus. The base vaccinia virus strain modified as described herein optionally contains one or more mutations compared to its parent strain, such as, but not limited to, one or more of the following: a deletion in TK (also referred to herein as "TK-") and a deletion in A52R (also referred to herein as "A52R-"). The vaccinia virus is optionally recombinant or selected to have low toxicity and accumulate in target tissues. In some embodiments, the modification in the viral backbone / viral genome is a modification that renders the vaccinia virus non-replicative or has reduced replicative capacity. Non-limiting examples of such modifications include mutations in the following viral genes: A1, A2, VH1, A33, and I7. In some embodiments, the viral backbone mutation is selected from the group consisting of a complete or partial deletion of the A52R gene, a complete or partial deletion of the TK gene, a complete or partial deletion of the B15R gene, a complete or partial deletion of the K7R gene, a complete or partial deletion of the B14R gene, a complete or partial deletion of the N1L gene, a complete or partial deletion of the K1L gene, a complete or partial deletion of the M2L gene, a complete or partial deletion of the A49R gene, a complete or partial deletion of the VH1 gene, a complete or partial deletion of the A33 gene, a complete or partial deletion of the A1 gene, a complete or partial deletion of the A2 gene, a complete or partial deletion of the I7 gene, and a complete or partial deletion of the A46R gene. As used herein, reference to a viral gene refers to the protein encoded by the gene (e.g., the A33 gene refers to the gene encoding the A33 protein).In some embodiments, viral backbone mutations, including any combination of substitutions, insertions, and deletions, result in sequences with less than 100%, less than 99%, less than 98%, less than 97%, less than 96%, less than 95%, less than 94%, less than 93%, less than 92%, less than 91%, less than 90%, or less sequence homology to the wild-type sequence of the viral gene or viral protein encoded by the gene. In some embodiments, the viral gene and the protein encoded thereby are selected from the group consisting of B15R, K7R, B14R, N1L, K1L, M2L, A49R, VH1, A33, A1, A2, I7, and A46R. In some embodiments, the viral backbone contains one, two, three, four, five, or more mutations in the amino acid sequence of the viral protein (e.g., viral antigen). In some examples, the viral antigen is selected from the group consisting of B15R, K7R, B14R, N1L, K1L, M2L, A49R, VH1, A33, A1, A2, I7, and A46R. In some embodiments, the present disclosure provides a recombinant vaccinia virus containing one or more mutations in the viral genome (viral backbone) such that the mutations increase the immune response of the T cell arm. The mutations can be additions, deletions, or substitutions of one or more nucleic acids in the viral genome (wild-type or attenuated native strain vaccinia virus). In a non-limiting example, the mutation is a complete or partial deletion of a gene known to inhibit cytokines involved in Th1 immune responses. As a non-limiting example, the mutation is a deletion of nucleic acids encoding B8R (an interferon gamma (IFN-g) binding protein) or C12L (an interleukin-18 (IL-18) binding protein). In a further non-limiting example, the mutation is a complete or partial deletion of a gene in innate immune signaling. Non-limiting examples of mutations include deletion of nucleic acids encoding B18R (a type I interferon (IFN) binding protein), A52R (a nuclear factor kappa B (NF-κB) inhibitor protein), E3L (a protein kinase (PKR) inhibitor), C4, and C16 (STING pathway inhibitors).

[0108] The oncolytic virus comprises one or more additional insertions or partial insertions of exogenous nucleic acids encoding one or more proteins, as described herein. In some embodiments, the one or more proteins comprise a chemokine receptor or a functional variant thereof, a TGF-beta inhibitor or a functional variant thereof, or interleukin-12 or a functional variant thereof. In some embodiments, the one or more proteins comprise a TGF-beta inhibitor or a functional variant thereof and interleukin-12 or a functional variant thereof. Exemplary chemokine receptors for inclusion include, without limitation, wild-type and / or mutant CXCR3, CXCR4, CCR2, or CCL2. The vaccinia virus of the present disclosure further comprises one or more additional deletions or partial deletions of one or more genes from A52R, B15R, K7R, A46R, N1L, E3L, K1L, M2L, C16, N2R, B8R, B18R, VH1, and functional domains, fragments, or variants thereof, or any combination thereof. In some cases, the vaccinia viruses provided herein comprise a complete or partial deletion of at least one of the A52R or TK viral genes and an insertion of exogenous nucleic acid encoding one or more proteins (e.g., one or more immunomodulator proteins).

[0109] In some embodiments, the oncolytic virus is a modified oncolytic virus that has one or more modifications that result in a superior therapeutic effect on tumor cells compared to an otherwise identical virus that does not contain the modifications. In some non-limiting examples, the superior therapeutic effect includes enhanced immune evasion of the virus, enhanced tumor-targeted systemic delivery of the virus, enhanced intratumor and intertumor spread of the virus, and enhanced tumor-specific replication of the virus, or release of immune modulators and antitumor agents into the extracellular matrix, or any combination thereof. In some cases, the modified oncolytic virus of the present disclosure is used as a platform vector for systemic delivery.

[0110] The oncolytic virus described herein comprises the exogenous nucleic acid described herein.In some embodiments, the oncolytic virus provided herein comprises the complete or partial deletion of TK gene and the insertion of the region encoding at least one of transforming growth factor-beta inhibitor and cytokine, such as IL-12.The exemplary sequence for integration has been described herein above.

[0111] In some embodiments, the oncolytic viruses provided herein comprise a complete or partial deletion of the A52R gene and an insertion of a region encoding a chemokine receptor. In some embodiments, the chemokine receptor comprises CXCR3. In some embodiments, the region encoding a chemokine receptor comprises a sequence selected from Table 7. In some embodiments, the promoter driving expression of the chemokine receptor is an early promoter, a late promoter, a strong early promoter, a weak early promoter, a strong late promoter, a weak late promoter, or any combination thereof. In some embodiments, the A52R promoter drives expression of the chemokine receptor. In some embodiments, the A52R promoter drives expression of the region encoding CXCR3.

[0112] In some embodiments of the present disclosure, modified oncolytic viruses are provided that include modifications that enhance tumor-targeted systemic delivery of the virus. Typically, oncolytic viruses are either (a) administered systemically, (b) inoculated locally into tumors, or (c) injected directly into tumors ("intratumoral delivery"). In some embodiments, systemic delivery of oncolytic viruses offers the opportunity to simultaneously treat both the primary tumor and any visible or undiagnosed metastatic deposits. As a result, this delivery method is a very attractive treatment option for patients with advanced / metastatic disease or inaccessible diseases, such as pancreatic cancer or brain cancer, where access is difficult due to physiological barriers, such as the blood-brain barrier. However, there are obstacles to the successful systemic delivery of many oncolytic viruses. For example, as described above, in some cases, host defenses limit the ability of most oncolytic viruses to infect tumors after systemic administration. Non-specific uptake by blood cells, complement, antibodies, and antiviral cytokines, as well as other tissues such as the lungs, liver, and spleen, tissue-resident macrophages, and poor viral escape from the vascular compartment, among others, are major barriers to systemic delivery of oncolytic viruses. In some embodiments of the present disclosure, the disclosed oncolytic viruses include modifications that promote persistent viral presence in the circulatory system through at least enhanced immune evasion, as described above. Alternatively, enhanced tumor-targeted delivery of viruses is desirable in certain circumstances because it not only increases therapeutic efficacy against cancer, but also alleviates safety concerns associated with virus-mediated tumor therapy by limiting non-tumor infection and avoiding undesirable side effects of viral infection. Certain embodiments herein relate to oncolytic viruses that include modifications that promote tumor-targeted delivery of viruses.

[0113] In some embodiments of the present disclosure, modified oncolytic viruses are provided that include modifications that enhance intratumoral and intertumoral spread of the virus. Enhanced intratumoral and intertumoral spread of oncolytic viruses is an effective way to boost therapeutic efficacy by increasing the number of cancer cells infected by the virus. In some embodiments, provided herein are modified oncolytic viruses that include exogenous nucleic acids. In some embodiments, provided herein are modified oncolytic viruses that include modifications in the viral genome. In some embodiments, provided herein are modified oncolytic viruses that include exogenous nucleic acids and modifications in the viral genome.

[0114] In some embodiments, oncolytic viruses include, but are not limited to: (i) viruses that naturally replicate preferentially in cancer cells and are non-pathogenic in humans, often due to their high susceptibility to natural antiviral signal transduction or dependence on oncogenic signal transduction pathways; and (ii) viruses that are genetically engineered for use.In some embodiments, oncolytic viruses are measles viruses, polioviruses, poxviruses, vaccinia viruses, adenoviruses, adeno-associated viruses, herpes simplex viruses, vesicular stomatitis viruses, reoviruses, Newcastle disease viruses, Seneca viruses, retroviruses, mengoviruses, or myxoma viruses.In certain embodiments, oncolytic viruses are poxviruses.In certain embodiments, oncolytic viruses are vaccinia viruses.

[0115] In some embodiments, modified oncolytic viruses are utilized. Generally, such viruses include modifications to components thereof, such as, but not limited to, modifications in the native genome ("backbone") of the virus, such as mutations or deletions of viral genes, introduction of exogenous nucleic acid, chemical modification of viral nucleic acid or viral proteins, and introduction of exogenous or modified viral proteins into the viral capsid.

[0116] In some embodiments, the modified oncolytic virus comprises a mutation or deletion in the TK gene and further comprises an exogenous nucleic acid encoding a TGF-beta inhibitor. In some embodiments, the modified oncolytic virus comprises a mutation or deletion in the TK gene and further comprises an exogenous nucleic acid encoding a TGF-beta inhibitor and an exogenous nucleic acid encoding a cytokine, e.g., IL-12. In some embodiments, the exogenous nucleic acid encoding IL-12 is inserted 5' to the exogenous nucleic acid encoding the TGF-beta inhibitor. In some embodiments, the exogenous nucleic acid encoding the TGF-beta inhibitor is inserted 5' to the exogenous nucleic acid encoding the IL-12.

[0117] In some embodiments, the modified oncolytic virus comprises a mutation or deletion of the A52R gene and further comprises an exogenous nucleic acid encoding a CXCR3 receptor. In some embodiments, the modified oncolytic virus comprises a mutation or deletion of the A52R gene (the A52R promoter is maintained) and further comprises an exogenous nucleic acid encoding a CXCR3 receptor.

[0118] In some embodiments, in modified oncolytic viruses, such as oncolytic vaccinia viruses, the viral TK gene is replaced with the TK gene from herpes simplex virus (HSV-TK). HSV TK optionally serves as a substitute for the deleted TK, providing multiple benefits. For example, in some embodiments, HSV TK is used as an additional therapeutic prodrug-converting enzyme to convert ganciclovir (GCV) to its cytotoxic metabolite in tumors. In addition to the added therapeutic effect, this modification also functions as a suicide gene, effectively killing vaccinia-expressing cells through the addition of GCV, thereby halting the virus in the event of an adverse event or uncontrolled replication. Thus, in some cases, the modified oncolytic viruses of the present disclosure act as a safety switch. In a further example, a mutant version of HSV TK is used to enable PET imaging of labeled substrates with significantly increased sensitivity. Thus, in some cases, modified oncolytic viruses containing HSV TK used in PET imaging can act as reporters of in vivo viral replication to determine therapeutic activity early after treatment.

[0119] In some cases, modified oncolytic virus comprises the above-mentioned full-length viral backbone gene or viral backbone protein, or a shortened version thereof, or a functional domain thereof, or a fragment thereof, or a variant thereof.In various examples, modified oncolytic virus comprises one or more mutations or deletions of viral backbone gene or viral backbone protein as described above.Mutation of viral backbone gene and viral backbone protein comprises insertion, deletion, substitution or modification of amino acid in nucleic acid sequence and protein sequence.In some cases, deletion comprises complete or partial deletion of viral backbone gene or protein.

[0120] In some embodiments, the modification of the oncolytic virus increases the efficacy of tumor-targeted systemic delivery of the virus by at least about 1.1 fold, 1.2 fold, 1.5 fold, 1.8 fold, 2 fold, 2.2 fold, 2.5 fold, 2.8 fold, 3 fold, 3.2 fold, 3.5 fold, 3.8 fold, 4 fold, 4.2 fold, 4.5 fold, 4.8 fold, 5 fold, 5.2 fold, 5.5 fold, 5.8 fold, 6 fold, 6.2 fold, 6.5 fold, 6.8 fold, 7 fold, 8 fold, 9 fold, 10 fold, 11 fold, 12 fold, 13 fold, 14 fold, 15 fold, 16 fold, 17 fold, 18 fold, 19 fold, 20 fold, 21 fold, 22 fold, 23 fold, 24 fold, 25 fold, 26 fold, 27 fold, 28 fold, 29 fold, 30 fold, 31 fold, 32 fold, 33 fold, 34 fold, 35 fold, 36 fold, 37 fold, 38 fold, 39 fold, 40 fold, 41 fold, 42 fold, 43 fold, 44 fold, 45 fold, 46 fold, 47 fold, 48 fold, 49 fold, 50 fold, 51 fold, 52 fold, 53 fold, 54 fold, 55 fold, 56 fold, 57 fold, 58 fold, 59 fold, 60 fold, 6 x, 7.2x, 7.5x, 7.8x, 8x, 8.2x, 8.5x, 8.8x, 9x, 9.2x, 9.5x, 9.8x, 10x, 12x, 14x, 15x, 16x, 18x, 20x, 25x, 30x, 35x, 40x 100x 4 times, 2.5 x 10 4 Double, 5 x 10 4 times, 7.5 x 10 4 times, 2.5 x 10 5 Double, 5 x 10 5 times, 7.5 x 10 5 double, 10 6 times, 2.5 x 10 6 Double, 5 x 10 6 times, 7.5 x 10 6 double, 10 7 times, 2.5 x 10 7 Double, 5 x 10 7 times, 7.5 x 10 7 double, 10 8 times, 2.5 x 10 8 Double, 5 x 10 8 times, 7.5 x 10 8 double, 10 9 times, 2.5 x 10 9 Double, 5 x 10 9 times, 7.5 x 10 9 double, 10 10In certain embodiments, the efficacy of tumor-targeted systemic delivery of a virus is measured by quantifying the virus that infects tumor cells, optionally compared with the virus that infects non-tumor cells in the body. For example, in some cases, virus quantification is performed by staining viral particles in tissue sections or blood smears in the case of leukemia, lymphoma, or myeloma. In some cases, such quantification is performed by using reporter molecules, such as luciferase and fluorescent proteins, engineered to be expressed by the virus. In some cases, such quantification is performed by quantifying viral genomes in tumors. It is also possible to measure tumor-targeted systemic delivery of a virus by quantifying certain downstream effects of viral infection in tumor cells, such as, but not limited to, cytokines in response to viral infection or lymphocyte accumulation. In some embodiments, the oncolytic virus comprises an exogenous nucleic acid encoding CXCR3, CXCR4, CCR2, or any combination thereof. In some embodiments, the presence of exogenous nucleic acid results in an approximately 5-10 fold increase in the efficacy of tumor-targeted systemic delivery of the virus compared to an otherwise identical oncolytic virus lacking the exogenous nucleic acid.

[0121] In some embodiments, the present invention provides a modified oncolytic virus comprising an exogenous nucleic acid encoding a chemokine receptor, and the forced expression of the chemokine receptor by the modified oncolytic virus results in a boosted immune response against the infected tumor. After infecting a tumor, the modified oncolytic virus replicates in tumor cells, resulting in the expression of chemokine receptors on the surface of the tumor cells. These membrane receptors act as decoy receptors, binding to and sequestering immunosuppressive chemokines (e.g., CXCL12 and / or CCL2) within the tumor. As a result, the immunosuppressive microenvironment in the tumor is altered, resulting in enhanced immunotherapeutic activity of the modified oncolytic virus compared to an identical virus that does not contain a nucleic acid encoding a chemokine receptor. In some embodiments, the increase in immunotherapeutic activity is at least about 1.1 fold, 1.1 fold, 1.2 fold, 1.5 fold, 1.8 fold, 2 fold, 2.2 fold, 2.5 fold, 2.8 fold, 3 fold, 3.2 fold, 3.5 fold, 3.8 fold, 4 fold, 4.2 fold, 4.5 fold, 4.8 fold, 5 fold, 5.2 fold, 5.5 fold, 5.8 fold, 6 fold, 6.2 fold, 6.5 fold, 6.8 fold, 7 fold, 7.2 fold, 7.5 fold, 7.8 fold, 8 fold, 8.2 fold , 8.5x, 8.8x, 9x, 9.2x, 9.5x, 9.8x, 10x, 12x, 14x, 15x, 16x, 18x, 20x, 25x, 30x, 35x, 40x, 45x, 50x, 55 100x 4 times, 2.5 x 10 4 Double, 5 x 10 4 times, 7.5 x 10 4 times, 2.5 x 10 5 Double, 5 x 10 5 double, 10 62-fold or even higher. Increased immunotherapeutic activity is reflected, but not limited to, by increased B cell accumulation in tumors, increased T cell responses to tumor-associated immunogens, or both. B cell accumulation is measured, for example, by quantifying B cells in tumors, and T cell immune activity is measured, for example, by interferon-γ (interferon-gamma) secretion in an ELISPOT assay.

[0122] In some embodiments, provided herein are modified oncolytic viruses comprising an exogenous nucleic acid encoding a chemokine receptor, wherein forced expression of the chemokine receptor by the modified oncolytic virus results in increased replication of the virus in tumor cells compared to an identical virus that does not comprise a nucleic acid encoding a chemokine receptor. In some embodiments, the modified oncolytic virus comprises an exogenous CXCR3-expressing nucleic acid. In some embodiments, the modified oncolytic virus comprises an exogenous CCR2-expressing nucleic acid, which increases tumor-specific replication of the virus. In some embodiments, the modified oncolytic virus comprises an exogenous CCR5-expressing nucleic acid, which increases tumor-specific replication of the virus. In some embodiments, the increase in tumor-specific replication is at least about 1.1 fold, 1.1 fold, 1.2 fold, 1.5 fold, 1.8 fold, 2 fold, 2.2 fold, 2.5 fold, 2.8 fold, 3 fold, 3.2 fold, 3.5 fold, 3.8 fold, 4 fold, 4.2 fold, 4.5 fold, 4.8 fold, 5 fold, 5.2 fold, 5.5 fold, 5.8 fold, 6 fold, 6.2 fold, 6.5 fold, 6.8 fold, 7 fold, 7.2 fold, 7.5 fold, 7.8 fold, 8 fold, 8.2 fold, 9 fold, 10 fold, 11 fold, 12 fold, 13 fold, 14 fold, 15 fold, 16 fold, 17 fold, 18 fold, 19 fold, 20 fold, 21 fold, 22 fold, 23 fold, 24 fold, 25 fold, 26 fold, 27 fold, 28 fold, 29 fold, 30 fold, 31 fold, 32 fold, 33 fold, 34 fold, 35 fold, 36 fold, 37 fold, 38 fold, 39 fold, 40 fold, 41 fold, 42 fold, 43 fold, 44 fold, 45 fold, 46 fold, 47 fold, 48 fold, 49 fold, 50 fold, 51 fold, 52 fold, 53 fold, 54 fold, 55 fold, 56 fold, 57 fold, x, 8.5x, 8.8x, 9x, 9.2x, 9.5x, 9.8x, 10x, 12x, 14x, 15x, 16x, 18x, 20x, 25x, 30x, 35x, 40x, 45x, 50x, 55x 100x 4 times, 2.5 x 10 4 Double, 5 x 10 4 times, 7.5 x 10 4 times, 2.5 x 10 5 Double, 5 x 105 double, 10 6 Exemplary methods for measuring increased viral delivery and spread in tumors include, but are not limited to, fluorescence- or bioluminescence-based imaging of reporter gene expression, quantitative PCR for detection of tumor concentrations of viral genomes, or plaque determination of plaque-forming units, or immunohistochemistry of viral proteins.

[0123] In some embodiments, the modified oncolytic virus comprises an exogenous nucleic acid encoding a chemokine receptor that is a chimeric protein. At least a portion of its extracellular domain is derived from a chemokine receptor that promotes tumor-targeted delivery of the virus, and at least a portion of its intracellular domain is derived from a chemokine receptor that promotes tumor-specific replication, inhibits immunosuppressive activity, or provides some other beneficial effect, or vice versa. For example, the modified oncolytic virus comprises a nucleic acid encoding a protein having the intracellular GTPase domain of CCR5 or CXCR3 and the extracellular chemokine-binding domain of CXCR4 or CCR2. In some cases, combining domains with different functionalities further improves the therapeutic performance of the modified oncolytic virus. In one embodiment of the present disclosure, the modified oncolytic virus comprises an exogenous nucleic acid encoding at least one chemokine receptor. In some cases, the modified oncolytic virus comprises an exogenous nucleic acid encoding two or more different chemokine receptors that are simultaneously expressed by the virus. Exemplary chemokine receptors that can be co-expressed from the modified oncolytic viruses described herein include CXCR4 and CCR2. In modified oncolytic viruses that express more than one chemokine receptor, a combined or synergistic effect on tumor cells is achieved for therapeutic applications of the oncolytic virus.

[0124] Treatment conditions Provided herein are methods for the treatment of cancer, comprising administering a composition described herein. In some embodiments, the method of treatment is for a hyperproliferative disease. In some embodiments, the hyperproliferative disease is cancer. In some embodiments, the hyperproliferative disease comprises a tumor. Treatments are contemplated that include delivery of a modified oncolytic virus, such as an oncolytic vaccinia virus, described herein. In some embodiments, the cancer is melanoma, hepatocellular carcinoma, breast cancer, lung cancer, peritoneal cancer, prostate cancer, bladder cancer, ovarian cancer, leukemia, lymphoma, kidney cancer, pancreatic cancer, epithelial carcinoma, gastric cancer, colon cancer, duodenal cancer, pancreatic adenocarcinoma, mesothelioma, glioblastoma multiforme, astrocytoma, multiple myeloma, prostate cancer, hepatocellular carcinoma, cholangiosarcoma, pancreatic adenocarcinoma, head and neck squamous cell carcinoma, colorectal cancer, gastrointestinal-type gastric adenocarcinoma, cervical squamous-cell carcinoma, osteosarcoma, epithelial ovarian cancer, acute lymphoblastic lymphoma, myeloproliferative neoplasm, or sarcoma.

[0125] In some embodiments, the compositions described herein are administered to cancer cells originating from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gums, head, kidney, liver, lung, nasopharynx, cervix, ovary, prostate, skin, stomach, testicle, tongue, or uterus. In addition, the cancer may optionally be of the following histological types, including, but not limited to, malignant neoplasm, carcinoma, undifferentiated carcinoma, giant cell and spindle cell carcinoma, small cell carcinoma, papillary carcinoma, squamous cell carcinoma, lymphoepithelial carcinoma, basal cell carcinoma, pilomatrix carcinoma, transitional cell carcinoma, papillary transitional cell carcinoma, adenocarcinoma, malignant gastrinoma, cholangiocarcinoma, hepatocellular carcinoma, combined hepatocellular carcinoma and cholangiocarcinoma, trabecular adenocarcinoma, adenoid cystic carcinoma, adenocarcinoma in adenomatous polyps, familial polyposis coli adenocarcinoma, solid tumors, malignant carcinoid tumor, bronchiolo-alveolar adenocarcinoma, papillary adenocarcinoma, chromophobe carcinoma, eosinophilic carcinoma, oxyphilic adenocarcinoma adenocarcinoma), basophilic carcinoma, clear cell adenocarcinoma, granular cell carcinoma, follicular adenocarcinoma, papillary and follicular adenocarcinoma, nonencapsulating sclerosing carcinoma, adrenocortical carcinoma, endometroid carcinoma, skin appendage carcinoma, apocrine adenocarcinoma, sebaceous carcinoma, auditory canal adenocarcinoma (ceruminous)adenocarcinoma, mucoepidermoid carcinoma, cystadenocarcinoma, papillary cystadenocarcinoma, papillary serous cystadenocarcinoma, mucinous cystadenocarcinoma, mucinous adenocarcinoma, signet ring cell carcinoma, invasive ductal carcinoma of the breast, medullary carcinoma, lobular carcinoma, inflammatory carcinoma, Paget's disease of the breast, pancreatic acinar cell carcinoma, adenosquamous carcinoma, adenocarcinoma with squamous metaplasia, malignant thymoma, malignant ovarian stromal tumor, malignant thecoma, malignant granulosa cell tumor, malignant androblastoma, Sertoli cell carcinoma, malignant Leydig cell tumor, malignant lipid cell tumor, malignant paraganglioma, malignant extramammary paraganglioma, pheochromocytoma, glomus angiosarcoma, malignant melanoma, amelanotic melanoma, superficial spreading melanoma, malignant melanoma in giant pigmented nevus , epithelioid cell melanoma, malignant blue nevus, sarcoma, fibrosarcoma, malignant fibrous histiocytoma, myxosarcoma, liposarcoma, leiomyosarcoma, rhabdomyosarcoma, embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, stromal sarcoma, malignant mixed tumor, mixed Müllerian tumor, nephroblastoma, hepatoblastoma, carcinosarcoma, malignant mesenchymoma, malignant Brenner tumor, malignant phyllodes tumor, synovial sarcoma, malignant mesothelioma, dysgerminoma, embryonal carcinoma, malignant teratoma, malignant ovarian stromal tumor, choriocarcinoma, malignant mesonephroma, angiosarcoma, malignant hemangioendothelioma, Kaposi's sarcoma, malignant hemangiopericytoma, lymphangiosarcoma, osteosarcoma, parosteal osteosarcoma, chondrosarcoma, malignant chondroblastoma, mesenchymal chondrosarcoma, giant cell tumor of bone, Ewing's sarcoma, malignant odontogenic tumor, ameloblastic odontogenic sarcoma odontosarcoma, malignant ameloblastoma, ameloblastic fibrosarcoma, malignant pinealoma, chordoma, malignant glioma, ependymoma, astrocytoma, protoplasmic astrocytoma, fibrous astrocytoma, astroblastoma, glioblastoma, oligodendroglioma, oligodendroblastoma, primitive neuroectodermal tumorneuroectodermal, cerebellar sarcoma, ganglioneuroblastoma, neuroblastoma, retinoblastoma, olfactory neurogenic tumor, malignant meningioma, neurofibrosarcoma, malignant schwannoma, malignant granular cell tumor, malignant lymphoma, Hodgkin's disease, Hodgkin's lymphoma, paragranuloma, malignant small lymphocytic lymphoma, malignant large cell diffuse lymphoma, malignant follicular lymphoma, mycosis fungoides, other specified non-Hodgkin's lymphoma, malignant histiocytosis, multiple myeloma, mast cell sarcoma, immunoproliferative small intestinal disease, leukemia, lymphoid leukemia, plasma cell leukemia, erythroleukemia, lymphosarcoma cell leukemia leukemia), myeloid leukemia, basophilic leukemia, eosinophilic leukemia, monocytic leukemia, mast cell leukemia, megakaryoblastic leukemia, myeloid sarcoma, or hairy cell leukemia. In some cases, metastatic solid cancers are treated using the modified oncolytic viruses of the present disclosure, e.g., modified oncolytic vaccinia viruses that are advantageous for systemic delivery. In some cases, solid cancers that are inaccessible or difficult to access, such as for intratumoral delivery of therapeutic agents, are treated using the modified oncolytic viruses of the present disclosure, e.g., modified oncolytic vaccinia viruses that are advantageous for systemic delivery. In some embodiments, the compositions described herein are used to treat cancers associated with increased expression of free fatty acids.

[0126] The present disclosure also contemplates a method for inhibiting or preventing the local invasiveness or metastasis, or both, of any type of primary cancer. In exemplary embodiments, the primary cancer is melanoma, non-small cell lung, small cell lung, lung, hepatocellular carcinoma, retinoblastoma, astrocytoma, glioblastoma, gum, tongue, leukemia, neuroblastoma, head, neck, breast, pancreas, prostate, kidney, bone, testis, ovary, mesothelioma, cervix, gastrointestinal tract, lymphoma, brain, colon, or bladder. In certain embodiments, the primary cancer is lung cancer. For example, the lung cancer is non-small cell lung cancer. Furthermore, the present disclosure can be used to prevent cancer or treat pre-cancerous or pre-malignant cells, including metaplasia, dysplasia, and hyperplasia, as needed. It can also be used to inhibit unwanted but benign cells, such as squamous cell metaplasia, dysplasia, benign prostatic hyperplasia, hyperplastic lesions, etc. In some embodiments, cancer or the progression to more severe forms of cancer is stopped, destroyed, or slowed by the methods of the present disclosure, including the modified oncolytic viruses discussed herein.

[0127] Furthermore, the modified oncolytic viruses disclosed herein are administered to treat tumors with high bioavailability of free fatty acids in the tumor microenvironment. In some cases, free fatty acids released by adipocytes in tumors in obese patients nourish and enhance the replication of the modified oncolytic viruses and the formation of EEV forms of the virus within the tumor. This benefit is also recognized in non-obese patients, particularly those with peritoneal cancer. For example, some peritoneal cancers are targets for therapy using the modified oncolytic viruses disclosed herein because they tend to grow in the omentum wall and are nourished by adipocytes, and as described above, the free fatty acids released by adipocytes in tumors nourish and enhance the replication of the modified oncolytic viruses within the tumor. The modified oncolytic viruses disclosed herein form increased titers of extracellular enveloped viruses (EEVs) in tumors with high bioavailability of free fatty acids.

[0128] Provided herein is a method for treating a subject by administering one or more modified oncolytic viruses disclosed herein.In this specification, "individual" or "subject" are used interchangeably to refer to human or non-human subjects.Non-limiting examples of non-human subjects include non-human primates, dogs, cats, mice, rats, guinea pigs, rabbits, pigs, poultry, horses, cows, goats, sheep, cetaceans, etc.In some embodiments, the subject is human.

[0129] Provided is a method for producing toxic effects in cancer cells, comprising administering a therapeutically effective amount of the modified virus described above, for example, an oncolytic vaccinia virus, or a pharmaceutical composition comprising the same to cancer cells.The present disclosure further provides a method for inhibiting at least one of the growth and proliferation of a second cancer cell, comprising administering the modified oncolytic virus described above to a first cancer cell so that the first cancer cell is infected with the virus.Therefore, in some embodiments of the method disclosed herein, it is contemplated that when a therapeutically effective amount of the oncolytic vaccinia virus described herein or a pharmaceutical composition comprising the same is administered, not all cancer or tumor cells are infected, and the growth of uninfected cells is inhibited without direct infection.

[0130] In some examples, the methods and compositions of the present disclosure are used to contact cancer cells or tumors with a therapeutically effective dose of the exemplary oncolytic vaccinia virus described herein or a pharmaceutical composition comprising the same, to induce tumor lysis, kill cells, inhibit growth, inhibit metastasis, reduce tumor size, and otherwise reverse or reduce the malignant phenotype of tumor cells.In certain embodiments, the effective amount of the modified oncolytic virus of the present disclosure, such as the oncolytic vaccinia virus described herein, or a pharmaceutical composition thereof, can comprise an amount sufficient to induce tumor lysis, destruction or lysis of cancer cells, or inhibit or reduce the growth or size of cancer cells.The reduction of cancer cell growth is indicated, for example, by cell death, or a slower replication rate or reduced growth rate of tumors comprising the cells, or the prolonged survival of subjects comprising cancer cells.

[0131] In some embodiments, use of the modified viruses described herein inhibits tumor growth by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or about 100% compared to an untreated tumor.

[0132] In some embodiments, use of the modified viruses described herein reduces tumor size by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or about 100% compared to an untreated tumor.

[0133] Provided herein is a method for treating a subject with cancer or a tumor, comprising administering to the subject an effective amount of the modified virus described above. In such methods, an effective amount includes an amount that reduces the growth rate or spread of the cancer or prolongs survival in the subject. The present disclosure provides a method for reducing tumor growth, comprising administering to a tumor an effective amount of the modified oncolytic virus described above. In certain embodiments, the effective amount of the modified virus or pharmaceutical composition thereof comprises an amount sufficient to induce slowing, inhibition, or reduction in tumor growth or size, including eradication of the tumor. Reduction of tumor growth is indicated, for example, by a reduced growth rate or prolonged survival of the tumor-bearing subject. In certain embodiments, the effective amount of the modified virus or pharmaceutical composition thereof comprises an amount sufficient to activate an anti-tumor response. In some embodiments, activating an anti-tumor response comprises activating T cells. In certain embodiments, the effective amount of the modified virus or pharmaceutical composition thereof comprises an amount sufficient to reduce the occurrence of tumor growth. In some embodiments, reducing the incidence of tumor growth includes inhibiting metastasis, preventing primary tumor growth, inhibiting existing tumor growth, or any combination thereof.

[0134] Provided herein is a method for determining the infectivity or anti-tumor activity of an oncolytic vaccinia virus described herein, or the amount of tumor-specific viral replication thereof, comprising the steps of: (i) administering to a subject a therapeutically effective amount of an oncolytic vaccinia virus or pharmaceutical composition according to the present disclosure that further expresses a luciferase reporter gene, alone or in combination with an additional therapy; (ii) collecting a first biological sample from the subject immediately after administering the virus and determining the level of the luciferase reporter in the first biological sample; (iii) collecting a second biological sample from the subject after administration in step (ii); and (iii) detecting the level of the luciferase reporter in the second biological sample; wherein if the level of luciferase is higher in step (iii) than in step (ii), the oncolytic vaccinia virus is determined to be infectious, exhibit anti-tumor activity, and exhibit tumor-specific viral replication. The second biological sample is collected at about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 15 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 1 month, or about 2 months after administration in step (i). In some embodiments, the above-described method in steps (i) and (iii) further comprises detecting the levels of one or more assayed cytokines, e.g., IL-2, IL-7, IL-8, IL-10, IFN-γ, GM-CSF, TNF-α, IL-6, IL-4, IL-5, and IL-13, in a plasma sample collected from the subject after administering to the subject a therapeutically effective amount of a modified oncolytic virus of the present disclosure, e.g., an oncolytic vaccinia virus described herein, or a pharmaceutical composition comprising the same.In some embodiments of the present disclosure, the increase in luciferase bioluminescence between steps (ii) and (iv) above is greater for the modified oncolytic viruses described herein compared to the same virus except that the modified oncolytic virus does not contain the modification. Other exemplary techniques for detecting and monitoring viral load after administration of the modified oncolytic virus include real-time quantitative PCR.

[0135] Provided herein is a method for monitoring pharmacokinetics after administration of a therapeutically effective amount of a modified oncolytic virus according to the present disclosure, such as an oncolytic vaccinia virus described herein or a pharmaceutical composition comprising a vaccinia virus. An exemplary method for monitoring pharmacokinetics includes the following steps: (i) administering to a subject a therapeutically effective amount of an oncolytic vaccinia virus or a pharmaceutical composition comprising the same, alone or in combination with an additional therapy; and (ii) measuring the pharmacokinetics at about 15 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 75 minutes, about 90 minutes, about 120 minutes, about 180 minutes, and about 240 minutes, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 15 hours, about 24 hours, about 30 minutes, about 45 minutes, about 60 minutes, about 75 minutes, about 90 minutes, about 120 minutes, about 180 minutes, and about 240 minutes after administration in step (i). The method includes (iii) collecting a biological sample from the subject at one or more time points selected from about 15 minutes, about 36 hours, about 48 hours, about 72 hours, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 1 month, and about 2 months later; and (iii) detecting the amount of viral genome (or a reporter gene inserted into the viral genome, e.g., luciferase) in the biological sample collected at the aforementioned time points. In some cases, the viral genome copies / mL are highest in the sample collected at 15 minutes, and further, the sample collected at 240 minutes does not contain a detectable amount of viral genome. Thus, in some cases, the viral peak is observed at about 15 minutes after administration, and the majority of the virus is cleared from the subject's system after about 240 minutes (or 4 hours). In some cases, a first viral peak is observed about 15 minutes after administration, and a second viral peak is observed in a biological sample taken at a subsequent time point, for example, about 30 minutes, about 45 minutes, about 60 minutes, or about 90 minutes. In an exemplary embodiment, the biological sample is blood, and the amount of viral genomes per mL is determined by quantitative PCR or other suitable technique.In some examples, a first viral peak is observed about 15 minutes after administration, and a second viral peak is observed about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 15 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 1 month, or up to about 2 months after administration of a modified oncolytic virus of the present disclosure, such as an oncolytic vaccinia virus described herein.

[0136] In some cases, tumor-selective replication of the modified virus, e.g., oncolytic vaccinia virus, is measured through the use of a reporter gene, e.g., a luciferase gene. In some embodiments, the luciferase gene is inserted into the genome of the virus, and tumor cells are infected with the virus. Bioluminescence in the infected tumor cells is measured to monitor tumor-selective replication. Some examples show an increase in luciferase reporter bioluminescence in the modified oncolytic virus of the present disclosure compared to the same oncolytic vaccinia virus that does not contain the modifications in the modified oncolytic virus.

[0137] The present invention provides a method for delivering the modified virus described herein.The modified virus provided herein is capable of increased replication in tumor cells compared to normal cells.In some embodiments, the modified virus produces about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 12 times, about 14 times, about 16 times, about 18 times, about 20 times, about 30 times, about 40 times, or about 50 times more copies per mg in tumor cells than in normal cells.

[0138] Provided herein are methods of contacting tumors with the modified viruses described herein. The modified viruses provided herein can increase the infiltration of CD3+CD8+ T cells in tumors compared to untreated tumors. In some embodiments, contacting tumors with the modified viruses described herein results in an increase of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% in CD3+CD8+ T cells in tumors compared to untreated tumors. In some embodiments, contacting tumors with the modified viruses described herein results in an increase of about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold in CD3+CD8+ T cells in tumors compared to untreated tumors.

[0139] Dosage In some embodiments, the amount of a modified oncolytic virus described herein administered to a subject is about 10 3 ~10 12 infectious viral particles or plaque-forming units (PFU), or approximately 10 5 ~10 10 PFU, or approximately 10 5 ~10 8 PFU, or approximately 10 8 ~10 10 In some embodiments, the amount of modified oncolytic virus of the present disclosure administered to a subject is about 10 PFU. 3 ~10 12 viral particles or plaque-forming units (PFU), or approximately 10 5 ~10 10 PFU, or approximately 10 5 ~10 8 PFU, or approximately 10 8 ~10 10 In some embodiments, the modified oncolytic viruses of the present disclosure are at about 10 PFU. 3 PFU / dose~about 10 4 PFU / dose, approximately 10 4 PFU / dose~about 10 5PFU / dose, approximately 10 5 PFU / dose~about 10 6 PFU / dose, approximately 10 7 PFU / dose~about 10 8 PFU / dose, approximately 10 9 PFU / dose~about 10 10 PFU / dose, approximately 10 10 PFU / dose~about 10 11 PFU / dose, approximately 10 11 PFU / dose~about 10 12 PFU / dose, approximately 10 12 PFU / dose~about 10 13 PFU / dose, approximately 10 13 PFU / dose~about 10 14 PFU / dose, or approximately 10 14 PFU / dose~about 10 15 In some embodiments, the modified oncolytic viruses of the present disclosure are administered at a dose comprising about 2 x 10 PFU / dose. 3 PFU / dose, 3×10 3 PFU / dose, 4×10 3 PFU / dose, 5×10 3 PFU / dose, 6×10 3 PFU / dose, 7×10 3 PFU / dose, 8×10 3 PFU / dose, 9×10 3 PFU / dose, approximately 10 4 PFU / dose, approximately 2 x 10 4 PFU / dose, approximately 3 x 10 4 PFU / dose, approximately 4 x 10 4 PFU / dose, approximately 5 x 10 4 PFU / dose, approximately 6 x 10 4 PFU / dose, approximately 7 x 10 4 PFU / dose, approximately 8 x 10 4 PFU / dose, approximately 9 x 10 4 PFU / dose, approximately 10 5 PFU / dose, 2×10 5 PFU / dose, 3×10 5 PFU / dose, 4×10 5 PFU / dose, 5×10 5 PFU / dose, 6×10 5 PFU / dose, 7×10 5PFU / dosage, 8×10 5 PFU / dosage, 9×10 5 PFU / dosage, approximately 10 6 PFU / dosage, approximately 2 × 10 6 PFU / dosage, approximately 3 x 10 6 PFU / dosage, approximately 4 x 10 6 PFU / dosage, approximately 5 x 10 6 PFU / dosage, approximately 6 x 10 6 PFU / dosage, approximately 7 x 10 6 PFU / dosage, approximately 8 x 10 6 PFU / dosage, approximately 9 x 10 6 PFU / dosage, approximately 10 7 PFU / dosage, approximately 2 × 10 7 PFU / dosage, approximately 3 x 10 7 PFU / dosage, approximately 4 x 10 7 PFU / dosage, approximately 5 x 10 7 PFU / dosage, approximately 6 x 10 7 PFU / dosage, approximately 7 x 10 7 PFU / dosage, approximately 8 x 10 7 PFU / dosage, approximately 9 x 10 7 PFU / dosage, approximately 10 8 PFU / dosage, approximately 2 × 10 8 PFU / dosage, approximately 3 x 10 8 PFU / dosage, approximately 4 x 10 8 PFU / dosage, approximately 5 x 10 8 PFU / dosage, approximately 6 x 10 8 PFU / dosage, approximately 7 x 10 8 PFU / dosage, approximately 8 x 10 8 PFU / dosage, approximately 9 x 10 8 PFU / dosage, approximately 10 9 PFU / dosage, approximately 2 × 10 9 PFU / dosage, approximately 3 x 10 9 PFU / dosage, approximately 4 x 10 9 PFU / dosage, approximately 5 x 10 9 PFU / dosage, approximately 6 x 10 9 PFU / dosage, approximately 7 x 10 9 PFU / dosage, approximately 8 x 10 9 PFU / dosage, approximately 9 x 10 9 PFU / dosage, approximately 10 10 PFU / dosage, approximately 2 × 10 10 PFU / dosage, approximately 3 x 1010 PFU / dose, approximately 4 x 10 10 PFU / dose, approximately 5 x 10 10 PFU / dose, approximately 6 x 10 10 PFU / dose, approximately 7 x 10 10 PFU / dose, approximately 8 x 10 10 PFU / dose, approximately 9 x 10 10 PFU / dose, approximately 10 10 PFU / dose, approximately 2 x 10 10 PFU / dose, approximately 3 x 10 10 PFU / dose, approximately 4 x 10 10 PFU / dose, approximately 5 x 10 10 PFU / dose, approximately 6 x 10 10 PFU / dose, approximately 7 x 10 10 PFU / dose, approximately 8 x 10 10 PFU / dose, approximately 9 x 10 10 PFU / dose, approximately 10 11 PFU / dose, approximately 2 x 10 11 PFU / dose, approximately 3 x 10 11 PFU / dose, approximately 4 x 10 11 PFU / dose, approximately 5 x 10 11 PFU / dose, approximately 6 x 10 11 PFU / dose, approximately 7 x 10 11 PFU / dose, approximately 8 x 10 11 PFU / dose, approximately 9 x 10 11 PFU / dose, or approximately 10 12 PFU / dose, approximately 10 12 PFU / dose~about 10 13 PFU / dose, approximately 10 13 PFU / dose~about 10 14 PFU / dose, or approximately 10 14 PFU / dose~about 10 15 In some embodiments, the modified oncolytic viruses of the present disclosure are administered at a dose comprising 5×10 PFU / dose. 9 In some embodiments, the modified oncolytic viruses of the present disclosure are administered in doses comprising up to 5×10 PFU / dose. 9 The dose is administered in a volume containing PFU / dose.

[0140] In some embodiments, the modified oncolytic viruses of the present disclosure are administered in an amount of about 103 Virus particles / dose ~ approx. 10 4 Viral particles / dose, approximately 10 4 Virus particles / dose ~ approx. 10 5 Viral particles / dose, approximately 10 5 Virus particles / dose ~ approx. 10 6 Viral particles / dose, approximately 10 7 Virus particles / dose ~ approx. 10 8 Viral particles / dose, approximately 10 9 Virus particles / dose ~ approx. 10 10 Viral particles / dose, approximately 10 10 Virus particles / dose ~ approx. 10 11 Viral particles / dose, approximately 10 11 Virus particles / dose ~ approx. 10 12 Viral particles / dose, approximately 10 12 Virus particles / dose ~ approx. 10 13 Viral particles / dose, approximately 10 13 Virus particles / dose ~ approx. 10 14 viral particles / dose, or approximately 10 14 Virus particles / dose ~ approx. 10 15 The dose containing the viral particles / dose is administered.

[0141] In some embodiments, the modified oncolytic viruses of the present disclosure are administered in an amount of about 10 3 PFU / kg ~ approx. 10 4 PFU / kg, approximately 10 4 PFU / kg ~ approx. 10 5 PFU / kg, approximately 10 5 PFU / kg ~ approx. 10 6 PFU / kg, approximately 10 7 PFU / kg ~ approx. 10 8 PFU / kg, approximately 10 9 PFU / kg ~ approx. 10 10 PFU / kg, approximately 10 10 PFU / kg ~ approx. 10 11 PFU / kg, approximately 10 11 PFU / kg ~ approx. 10 12 PFU / kg, approximately 10 12 PFU / kg ~ approx. 10 13 PFU / kg, approximately 10 13 PFU / kg ~ approx. 1014 PFU / kg, or approximately 10 14 PFU / kg ~ approx. 10 15 In some embodiments, the modified oncolytic virus of the present disclosure is administered at a dose comprising about 2 x 10 PFU / kg. 3 PFU / kg, 3 × 10 3 PFU / kg, 4 × 10 3 PFU / kg, 5 × 10 3 PFU / kg, 6 × 10 3 PFU / kg, 7 × 10 3 PFU / kg, 8 × 10 3 PFU / kg, 9 × 10 3 PFU / kg, approximately 10 4 PFU / kg, approximately 2×10 4 PFU / kg, approximately 3×10 4 PFU / kg, approximately 4×10 4 PFU / kg, approximately 5×10 4 PFU / kg, approximately 6×10 4 PFU / kg, approximately 7×10 4 PFU / kg, approximately 8×10 4 PFU / kg, approximately 9×10 4 PFU / kg, approximately 10 5 PFU / kg, 2 × 10 5 PFU / kg, 3 × 10 5 PFU / kg, 4 × 10 5 PFU / kg, 5 × 10 5 PFU / kg, 6 × 10 5 PFU / kg, 7 × 10 5 PFU / kg, 8 × 10 5 PFU / kg, 9 × 10 5 PFU / kg, approximately 10 6 PFU / kg, approximately 2×10 6 PFU / kg, approximately 3×10 6 PFU / kg, approximately 4×10 6 PFU / kg, approximately 5×10 6 PFU / kg, approximately 6×10 6 PFU / kg, approximately 7×10 6 PFU / kg, approximately 8×10 6 PFU / kg, approximately 9×10 6 PFU / kg, approximately 10 7 PFU / kg, approximately 2×107 PFU / kg, approximately 3×10 7 PFU / kg, approximately 4×10 7 PFU / kg, approximately 5 × 10 7 PFU / kg, approximately 6×10 7 PFU / kg, approximately 7×10 7 PFU / kg, approximately 8×10 7 PFU / kg, approximately 9×10 7 PFU / kg, approximately 10 8 PFU / kg, approximately 2×10 8 PFU / kg, approximately 3×10 8 PFU / kg, approximately 4×10 8 PFU / kg, approximately 5 × 10 8 PFU / kg, approximately 6×10 8 PFU / kg, approximately 7×10 8 PFU / kg, approximately 8×10 8 PFU / kg, approximately 9×10 8 PFU / kg, approximately 10 9 PFU / kg, approximately 2×10 9 PFU / kg, approximately 3×10 9 PFU / kg, approximately 4×10 9 PFU / kg, approximately 5 × 10 9 PFU / kg, approximately 6×10 9 PFU / kg, approximately 7×10 9 PFU / kg, approximately 8×10 9 PFU / kg, approximately 9×10 9 PFU / kg, approximately 10 10 PFU / kg, approximately 2×10 10 PFU / kg, approximately 3×10 10 PFU / kg, approximately 4×10 10 PFU / kg, approximately 5 × 10 10 PFU / kg, approximately 6×10 10 PFU / kg, approximately 7×10 10 PFU / kg, approximately 8×10 10 PFU / kg, approximately 9×10 10 PFU / kg, approximately 10 10 PFU / kg, approximately 2×10 10 PFU / kg, approximately 3×10 10 PFU / kg, approximately 4×10 10 PFU / kg, approximately 5 × 10 10 PFU / kg, approximately 6×10 10PFU / kg, approximately 7×10 10 PFU / kg, approximately 8×10 10 PFU / kg, approximately 9×10 10 PFU / kg, approximately 10 11 PFU / kg, approximately 2×10 11 PFU / kg, approximately 3×10 11 PFU / kg, approximately 4×10 11 PFU / kg, approximately 5×10 11 PFU / kg, approximately 6×10 11 PFU / kg, approximately 7×10 11 PFU / kg, approximately 8×10 11 PFU / kg, approximately 9×10 11 PFU / kg, or approximately 10 12 PFU / kg, approximately 10 12 PFU / kg ~ approx. 10 13 PFU / kg, approximately 10 13 PFU / kg ~ approx. 10 14 PFU / kg, or approximately 10 14 PFU / kg ~ approx. 10 15 In some embodiments, the modified oncolytic viruses of the present disclosure are administered at a dose comprising 5×10 PFU / kg. 9 In some embodiments, the modified oncolytic viruses of the present disclosure are administered at a dose comprising up to 5×10 PFU / kg. 9 The dose is administered in the amount of PFU / kg.

[0142] In some embodiments, the modified oncolytic viruses of the present disclosure are administered in an amount of about 10 3 Virus particles / kg ~ approx. 10 4 Virus particles / kg, approximately 10 4 Virus particles / kg ~ approx. 10 5 Virus particles / kg, approximately 10 5 Virus particles / kg ~ approx. 10 6 Virus particles / kg, approximately 10 7 Virus particles / kg ~ approx. 10 8 Virus particles / kg, approximately 10 9 Virus particles / kg ~ approx. 10 10 Virus particles / kg, approximately 10 10 Virus particles / kg ~ approx. 10 11 Virus particles / kg, approximately 1011 Virus particles / kg ~ approx. 10 12 Virus particles / kg, approximately 10 12 Virus particles / kg ~ approx. 10 13 Virus particles / kg, approximately 10 13 Virus particles / kg ~ approx. 10 14 virus particles / kg, or approximately 10 14 Virus particles / kg ~ approx. 10 15 It is administered in a dose containing viral particles / kg.

[0143] In certain embodiments, the liquid dosage forms of the oncolytic vaccinia viruses described herein contain about 10 3 PFU / mL~about 10 4 PFU / mL, approximately 10 4 PFU / mL~about 10 5 PFU / mL, approximately 10 5 PFU / mL~about 10 6 PFU / mL, approximately 10 7 PFU / mL~about 10 8 PFU / mL, approximately 10 9 PFU / mL~about 10 10 PFU / mL, approximately 10 10 PFU / mL~about 10 11 PFU / mL, approximately 10 11 PFU / mL~about 10 12 PFU / mL, approximately 10 12 PFU / mL~about 10 13 PFU / mL, approximately 10 13 PFU / mL~about 10 14 PFU / mL, or approximately 10 14 PFU / mL~about 10 15 In some embodiments, the modified oncolytic viruses of the present disclosure comprise a viral dose of about 2 x 10 PFU / mL. 3 PFU / mL, 3 × 10 3 PFU / mL, 4 × 10 3 PFU / mL, 5 × 10 3 PFU / mL, 6 × 10 3 PFU / mL, 7 × 10 3 PFU / mL, 8 × 10 3 PFU / mL, 9 × 10 3 PFU / mL, approximately 104 PFU / mL, approximately 2 × 10 4 PFU / mL, approximately 3 × 10 4 PFU / mL, approximately 4 × 10 4 PFU / mL, approximately 5 × 10 4 PFU / mL, approximately 6 × 10 4 PFU / mL, approximately 7 × 10 4 PFU / mL, approximately 8 × 10 4 PFU / mL, approximately 9 × 10 4 PFU / mL, approximately 10 5 PFU / mL, 2×10 5 PFU / mL, 3×10 5 PFU / mL, 4×10 5 PFU / mL, 5×10 5 PFU / mL, 6×10 5 PFU / mL, 7×10 5 PFU / mL, 8×10 5 PFU / mL, 9×10 5 PFU / mL, approximately 10 6 PFU / mL, approximately 2 × 10 6 PFU / mL, approximately 3 × 10 6 PFU / mL, approximately 4 × 10 6 PFU / mL, approximately 5 × 10 6 PFU / mL, approximately 6 × 10 6 PFU / mL, approximately 7 × 10 6 PFU / mL, approximately 8 × 10 6 PFU / mL, approximately 9 × 10 6 PFU / mL, approximately 10 7 PFU / mL, approximately 2 × 10 7 PFU / mL, approximately 3 × 10 7 PFU / mL, approximately 4 × 10 7 PFU / mL, approximately 5 × 10 7 PFU / mL, approximately 6 × 10 7 PFU / mL, approximately 7 × 10 7 PFU / mL, approximately 8 × 10 7 PFU / mL, approximately 9 × 10 7 PFU / mL, approximately 10 8 PFU / mL, approximately 2 × 10 8 PFU / mL, approximately 3 × 10 8 PFU / mL, approximately 4 × 10 8 PFU / mL, approximately 5 × 10 8PFU / mL, approximately 6 × 10 8 PFU / mL, approximately 7 × 10 8 PFU / mL, approximately 8 × 10 8 PFU / mL, approximately 9 × 10 8 PFU / mL, approximately 10 9 PFU / mL, approximately 2 × 10 9 PFU / mL, approximately 3 × 10 9 PFU / mL, approximately 4 × 10 9 PFU / mL, approximately 5 × 10 9 PFU / mL, approximately 6 × 10 9 PFU / mL, approximately 7 × 10 9 PFU / mL, approximately 8 × 10 9 PFU / mL, approximately 9 × 10 9 PFU / mL, approximately 10 10 PFU / mL, approximately 2 × 10 10 PFU / mL, approximately 3 × 10 10 PFU / mL, approximately 4 × 10 10 PFU / mL, approximately 5 × 10 10 PFU / mL, approximately 6 × 10 10 PFU / mL, approximately 7 × 10 10 PFU / mL, approximately 8 × 10 10 PFU / mL, approximately 9 × 10 10 PFU / mL, approximately 10 10 PFU / mL, approximately 2 × 10 10 PFU / mL, approximately 3 × 10 10 PFU / mL, approximately 4 × 10 10 PFU / mL, approximately 5 × 10 10 PFU / mL, approximately 6 × 10 10 PFU / mL, approximately 7 × 10 10 PFU / mL, approximately 8 × 10 10 PFU / mL, approximately 9 × 10 10 PFU / mL, approximately 10 11 PFU / mL, approximately 2 × 10 11 PFU / mL, approximately 3 × 10 11 PFU / mL, approximately 4 × 10 11 PFU / mL, approximately 5 × 10 11 PFU / mL, approximately 6 × 10 11 PFU / mL, approximately 7 × 10 11 PFU / mL, approximately 8 × 10 11 PFU / mL, approximately 9 × 10 11PFU / mL, or approximately 10 12 PFU / mL, approximately 10 12 PFU / mL~about 10 13 PFU / mL, approximately 10 13 PFU / mL~about 10 14 PFU / mL, or approximately 10 14 PFU / mL~about 10 15 In some embodiments, the modified oncolytic viruses of the present disclosure are administered at a dose comprising 5×10 PFU / mL. 9 In some embodiments, the modified oncolytic viruses of the present disclosure are administered at a dose comprising up to 5×10 PFU / mL. 9 It is administered in a dose containing PFU / mL.

[0144] In some cases, when the modified oncolytic virus is administered by injection, the dosage is about 10 per injection. 3 10 viral particles per injection 4 10 viral particles per injection 5 10 viral particles per injection 6 10 viral particles per injection 7 10 viral particles per injection 8 10 viral particles per injection 9 10 viral particles per injection 10 10 viral particles per injection 11 10 viral particles per injection 12 2 x 10 viral particles per injection 12 10 viral particles per injection 13 10 viral particles per injection 14 virus particles, or 10 per injection 15 In a further example, when the modified oncolytic virus is administered by injection, the dosage is about 10 viral particles per injection. 3 10 infectious viral particles per injection 4 10 infectious viral particles per injection 5 10 infectious viral particles per injection6 10 infectious viral particles per injection 7 10 infectious viral particles per injection 8 10 infectious viral particles per injection 9 10 infectious viral particles per injection 10 10 infectious viral particles per injection 11 10 infectious viral particles per injection 12 2 x 10 infectious viral particles per injection 12 10 infectious viral particles per injection 13 10 infectious viral particles per injection 14 infectious viral particles, or 10 per injection 15In certain embodiments, the virus is administered in an amount sufficient to induce tumor lysis in at least about 20% of the cells in the tumor, at least about 30% of the cells in the tumor, at least about 40% of the cells in the tumor, at least about 50% of the cells in the tumor, at least about 60% of the cells in the tumor, at least about 70% of the cells in the tumor, at least about 80% of the cells in the tumor, or at least about 90% of the cells in the tumor. In certain embodiments, a single dose of virus refers to the amount administered to a subject or tumor over a period of 1 hour, 2 hours, 5 hours, 10 hours, 15 hours, 20 hours, or 24 hours. In certain embodiments, the dose is spread over time or by separate injections. In certain embodiments, multiple doses (e.g., 2, 3, 4, 5, 6, or more doses) of vaccinia virus are administered to a subject, e.g., the second treatment occurs within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days, or within 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, or 7 weeks, of the first treatment. In certain embodiments, multiple doses of the modified oncolytic virus are administered to a subject over a period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or more, or 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or more. In certain embodiments, the oncolytic viruses or pharmaceutical compositions described herein are administered for about 1 week to about 2 weeks, about 2 weeks to about 3 weeks, about 3 weeks to about 4 weeks, about 4 weeks to about 5 weeks, about 6 weeks to about 7 weeks, about 7 weeks to about 8 weeks, about 8 weeks to about 9 weeks, about 9 weeks to about 10 weeks, about 10 weeks to about 11 weeks, about 11 weeks to about 12 weeks, about 12 weeks to about 24 weeks, about 24 weeks to about 48 weeks, about 48 weeks, or about 52 weeks, or longer.The administration frequency of the oncolytic vaccinia virus or pharmaceutical composition described herein is, in certain cases, once a day, twice a day, once a week, once every three weeks, once every four weeks (or once a month), once every eight weeks (or once every two months), once every 12 weeks (or once every three months), or once every 24 weeks (once every six months). In some embodiments of the methods disclosed herein, the oncolytic vaccinia virus or pharmaceutical composition is independently administered at an initial dose for a first period, at an intermediate dose for a second period, and at a high dose for a third period. In some embodiments, the initial dose is lower than the intermediate dose, and the intermediate dose is lower than the high dose. In some embodiments, the first, second, and third periods are independently about 1 week to about 2 weeks, about 2 weeks to about 3 weeks, about 3 weeks to about 4 weeks, about 4 weeks to about 5 weeks, about 6 weeks to about 7 weeks, about 7 weeks to about 8 weeks, about 8 weeks to about 9 weeks, about 9 weeks to about 10 weeks, about 10 weeks to about 11 weeks, about 11 weeks to about 12 weeks, about 12 weeks to about 24 weeks, about 24 weeks to about 48 weeks, about 48 weeks, or about 52 weeks, or longer.

[0145] In some examples, the subject is placed on a low-carbohydrate diet, e.g., a ketogenic diet, before, concurrently with, and after administration of a modified oncolytic virus described herein, e.g., an oncolytic vaccinia virus, or a pharmaceutical composition comprising same, according to any of the methods of treatment described herein. In certain embodiments, the subject is placed on a diet comprising consuming less than 500 grams of carbohydrates per day, less than 450 grams of carbohydrates per day, less than 450 grams of carbohydrates per day, less than 400 grams of carbohydrates per day, less than 350 grams of carbohydrates per day, less than 300 grams of carbohydrates per day, less than 250 grams of carbohydrates per day, less than 200 grams of carbohydrates per day, less than 150 grams of carbohydrates per day, less than 100 grams of carbohydrates per day, less than 90 grams of carbohydrates per day, less than 80 grams of carbohydrates per day, less than 70 grams of carbohydrates per day, less than 60 grams of carbohydrates per day, less than 50 grams of carbohydrates per day, less than 40 grams of carbohydrates per day, less than 30 grams of carbohydrates per day, less than 20 grams of carbohydrates per day, less than 10 grams of carbohydrates per day.

[0146] An exemplary method for delivering the modified oncolytic virus of the present disclosure, such as the oncolytic vaccinia virus described herein, or a pharmaceutical composition comprising the same to cancer or tumor cells is intratumoral injection. However, alternative administration methods, such as intravenous, infusion, parenteral, intravenous, intradermal, intramuscular, transdermal, rectal, intraurethral, ​​intravaginal, intranasal, intrathecal, or intraperitoneal, can also be used. The route of administration varies depending on the location and nature of the tumor. In certain embodiments, the route of administration is intraocular, transdermal, parenteral, intraperitoneal, intravenous, intramuscular, intranasal, subcutaneous, regional (e.g., near the tumor, particularly using the tumor vasculature or adjacent vasculature), percutaneous, intrathecal, intratracheal, intraperitoneal, intraarterial, intravesical, intratumoral, inhalation, perfusion, lavage, or oral. The injection dose of the oncolytic virus is administered as a bolus injection or slow infusion. In certain embodiments, modified oncolytic virus is administered to patient from a source implanted in patient.In certain embodiments, modified oncolytic virus is administered by continuous infusion over a selected period of time.In some cases, the oncolytic vaccinia virus described herein or the pharmaceutical composition comprising it is administered at a therapeutically effective dose by infusion over about 15 minutes, about 30 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 75 minutes, about 90 minutes, about 100 minutes, or about 120 minutes or longer. The oncolytic viruses or pharmaceutical compositions of the present disclosure are administered as a liquid dosage, wherein the total volume administered is about 1 mL to about 5 mL, about 5 mL to 10 mL, about 15 mL to about 20 mL, about 25 mL to about 30 mL, about 30 mL to about 50 mL, about 50 mL to about 100 mL, about 100 mL to 150 mL, about 150 mL to about 200 mL, about 200 mL to about 250 mL, about 250 mL to about 300 mL, about 300 mL to about 350 mL, about 350 mL to about 400 mL, about 400 mL to about 450 mL, about 450 mL to 500 mL, about 500 mL to 750 mL, or about 750 mL to 1000 mL.

[0147] formulation Pharmaceutical compositions comprising the modified viruses described herein, such as oncolytic vaccinia viruses, can be prepared as solutions, dispersions in glycerol, liquid polyethylene glycol, and any combination thereof in oil, solid dosage forms, inhalable dosage forms, intranasal dosage forms, liposomal formulations, nanoparticle dosage forms, microparticle dosage forms, polymeric dosage forms, or any combination thereof. In some embodiments, the pharmaceutical compositions described herein include stabilizers and buffers. In some embodiments, the pharmaceutical compositions described herein can include solubilizers, such as sterile water and Tris buffer. In some embodiments, the pharmaceutical compositions described herein can include additives. Non-limiting examples of suitable additives include buffering agents, preservatives, stabilizers, binders, compaction agents, lubricants, chelating agents, dispersion enhancers, disintegrants, flavoring agents, sweeteners, and coloring agents.

[0148] In certain embodiments, the buffering agent includes phosphate buffered saline (PBS), Dulbecco's PBS (DPBS), TRIS buffered saline (TBS), Hank's balanced salt solution (HBSS), Earl's balanced salt solution (EBSS), standard citrate saline (SSC), HEPES buffered saline (HBS), or Gey's balanced salt solution.

[0149] In certain embodiments, the pharmaceutical composition of the present disclosure comprises an effective amount of the modified virus disclosed herein in combination with a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable" includes any carrier that does not interfere with the effectiveness of the biological activity of the active ingredient and / or is not toxic to the patient to whom it is administered. Non-limiting examples of suitable pharmaceutical carriers include phosphate-buffered saline solution, water, emulsions, such as oil / water emulsions, various types of wetting agents, and sterile solutions. Additional non-limiting examples of pharmaceutically compatible carriers include gels, bioabsorbable matrix materials, implanted elements containing the modified oncolytic virus, or any other suitable vehicle, delivery, or dispensing means or material. Such carriers are formulated by conventional methods and administered to a subject in an effective amount.

[0150] Method of production The modified oncolytic virus of the present disclosure is produced by methods known to those skilled in the art. In certain embodiments, the modified oncolytic virus is propagated in suitable host cells, such as HeLa cells, 293 cells, or Vero cells, isolated from the host cells, and stored under conditions that promote the stability and integrity of the virus so that the decrease in infectivity over time is minimized. In certain exemplary methods, the modified oncolytic virus is propagated in host cells using cell stacks, roller bottles, or perfusion bioreactors. In some examples, downstream methods for purifying the modified oncolytic virus include filtration (e.g., depth filtration, tangential flow filtration, or a combination thereof), ultracentrifugation, chromatographic capture, or any combination thereof. The modified oncolytic virus is stored, for example, by freezing or drying, for example, by lyophilization. In certain embodiments, prior to administration, the stored modified oncolytic virus is reconstituted (if dried for storage) and diluted in a pharmaceutically acceptable carrier for administration.

[0151] Some embodiments provide that the modified oncolytic viruses described herein exhibit higher titers in HeLa cells and 293 cells compared to the same virus but without the modifications in the modified oncolytic virus. In certain cases, higher titers are found in HeLa cells and 293 cells for the modified oncolytic viruses.

[0152] kit In some embodiments, the present disclosure provides a kit for administering the modified oncolytic virus described herein. In certain embodiments, the kit of the present disclosure includes the above-mentioned modified oncolytic virus or a pharmaceutical composition containing the modified oncolytic virus. In certain embodiments, the kit of the present disclosure further includes one or more components, such as instructions for use, devices, and additional reagents, and components for carrying out the methods disclosed above, such as tubes, containers, and syringes. In certain embodiments, the kit of the present disclosure further includes one or more drugs, such as at least one of an anticancer agent, an immunomodulatory agent, or any combination thereof, to be administered in combination with the modified virus.

[0153] In certain embodiments, the kits of the present disclosure include one or more containers containing the modified viruses disclosed herein. For example, and without limitation, the kits of the present disclosure include one or more containers containing the modified oncolytic viruses of the present disclosure.

[0154] In certain embodiments, the kits of the present disclosure include instructions for use, a device for administering the modified oncolytic virus to a subject, or a device for administering an additional agent or compound to a subject. For example, and without limitation, the instructions include instructions for the modified oncolytic virus and other components optionally included in the kit, as well as instructions for administration, including methods for determining the appropriate condition of the subject for administering the modified virus, the appropriate dosage, and the appropriate administration method. The instructions also optionally include guidance for monitoring the subject over the treatment period.

[0155] In certain embodiments, the kit of the present disclosure includes a device for administering modified oncolytic viruses to a subject.Any of the various devices known in the art for administering medicaments and pharmaceutical compositions can be included in the kit provided herein.For example, but not limited to, such devices include hypodermic needles, intravenous needles, catheters, needleless injection devices, inhalers, and liquid dispensers, such as eye droppers.In certain embodiments, the modified oncolytic viruses to be delivered systemically, for example, by intravenous injection, intratumoral injection, or intraperitoneal injection, are included in the kit together with hypodermic needles and syringes.

[0156] While preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the embodiments of the present disclosure described herein may be utilized in practicing the disclosure. It is intended that the following claims define the scope of the disclosure, and that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0157] Illustrative Embodiments

[0003] Provided herein is a composition comprising a vector, the vector comprising an exogenous nucleic acid comprising a sequence encoding a cytokine or a functional variant thereof, an exogenous nucleic acid comprising a sequence encoding a chemokine receptor or a functional variant thereof, and a first promoter region upstream of the sequence encoding the chemokine receptor, the first promoter region causing expression of the chemokine receptor prior to expression of the cytokine.

[0004] Further provided herein is a composition, wherein the encoded chemokine receptor comprises at least one of a C-X receptor, a C-C receptor, a C-C receptor, an X-C receptor, a functional fragment thereof, a functional variant thereof, or any combination thereof. Further provided herein is a composition wherein the encoded chemokine receptor comprises at least one of CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, CX3CR1, XCR1, a functional fragment thereof, a functional variant thereof, or any combination thereof. Further provided herein is a composition wherein the encoded chemokine receptor is CXCR3. Further provided herein is a composition wherein the encoded chemokine receptor comprises an amino acid sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:42 or SEQ ID NO:43. Further provided herein is a composition wherein the encoded chemokine receptor comprises the amino acid sequence set forth in SEQ ID NO:42 or SEQ ID NO:43. Further provided herein is a composition wherein the first promoter region comprises an early promoter. Further provided herein are compositions wherein the early promoter comprises any one of A52R, pB8, mH5, I4L, LEO, pF11, I3L, P7.5, TK promoter, F7L, H5R, short synthetic promoter (SSP), or any variation or combination thereof. Further provided herein are compositions wherein the early promoter comprises the A52R promoter. Further provided herein are compositions wherein the encoded cytokine comprises IL-12 or a functional variant thereof.Further provided herein are compositions in which the encoded IL-12 is murine IL-12 or human IL-12. Further provided herein are compositions in which the encoded IL-12 comprises an alpha subunit and a beta subunit. Further provided herein are compositions in which the sequences encoding the IL-12 alpha subunit and the IL-12 beta subunit further comprise a sequence encoding a linker. Further provided herein are compositions in which the encoded IL-12 alpha subunit comprises an amino acid sequence having at least 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NO: 13 or SEQ ID NO: 16. Further provided herein are compositions in which the encoded IL-12 alpha subunit comprises an amino acid sequence set forth in any one of SEQ ID NO: 13 or SEQ ID NO: 16. Further provided herein are compositions in which the encoded IL-12 beta subunit comprises an amino acid sequence having at least 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NO: 14 or SEQ ID NO: 17. Further provided herein is a composition wherein the IL-12 beta subunit comprises the amino acid sequence set forth in any one of SEQ ID NO: 14 or SEQ ID NO: 17. Further provided herein is a composition wherein the encoded linker comprises an amino acid sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 18. Further provided herein is a composition wherein the encoded linker comprises the amino acid sequence set forth in SEQ ID NO: 18. Further provided herein is a composition wherein the encoded IL-12 comprises an amino acid sequence having at least 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NO: 12 or SEQ ID NO: 15. Further provided herein is a composition wherein the encoded IL-12 comprises an amino acid sequence set forth in any one of SEQ ID NO: 12 or SEQ ID NO: 15. Further provided herein is a composition wherein the sequence encoding IL-12 comprises a nucleic acid sequence having at least 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NO: 64 or SEQ ID NO: 66.Further provided herein is a composition wherein the sequence encoding IL-12 comprises the nucleic acid sequence set forth in any one of SEQ ID NOs: 64 or 66. Further provided herein is a composition further comprising an exogenous nucleic acid comprising a sequence encoding an inhibitor of transforming growth factor beta (TGF-beta) activity, wherein the first promoter region causes expression of a chemokine receptor prior to expression of the inhibitor of TGF-beta activity. Further provided herein is a composition wherein the encoded inhibitor of TGF-beta activity comprises a TGF-beta dominant negative, a TGF-beta receptor dominant negative, a protein that binds to TGF-beta, or a protein that binds to the TGF-beta receptor. Further provided herein is a composition wherein the encoded protein that binds to the TGF-beta receptor is a protein comprising a TGF-beta domain. Further provided herein is a composition wherein the encoded protein comprising a TGF-beta domain comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-9. Further provided herein is a composition wherein an encoded protein comprising a domain of TGF-beta comprises an amino acid sequence having at least 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NO:7 or SEQ ID NO:8. Further provided herein is a composition wherein an encoded protein comprising a domain of TGF-beta comprises an amino acid sequence set forth in any one of SEQ ID NO:7 or SEQ ID NO:8. Further provided herein is a composition wherein a sequence encoding an inhibitor of transforming growth factor beta (TGF-beta) activity comprises a nucleic acid sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:67. Further provided herein is a composition wherein a sequence encoding an inhibitor of TGF-beta activity comprises the nucleic acid sequence set forth in SEQ ID NO:67. Further provided herein is a composition wherein the protein that binds to the TGF-beta receptor is a TGF-beta fusion protein. Further provided herein is a composition wherein the encoded inhibitor of TGF-beta activity comprises a protein that binds to a TGF-beta receptor, and wherein the protein that binds to a TGF-beta receptor comprises a TGF-beta fusion 1 (TGFbf1) protein.Further provided herein is a composition wherein the TGFbf1 protein comprises a mouse IL-2 signal peptide and TGF-beta variant 1 (TGFbv1). Further provided herein is a composition wherein the mouse IL-2 signal peptide comprises an amino acid sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 36. Further provided herein is a composition wherein the mouse IL-2 signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 36. Further provided herein is a composition wherein the TGFbv1 comprises an amino acid sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 7. Further provided herein is a composition wherein the TGFbv1 comprises the sequence set forth in SEQ ID NO: 7. Further provided herein is a composition wherein the encoded TGF-beta activity inhibitor comprises a protein that binds to the TGF-beta receptor, and wherein the protein that binds to the TGF-beta receptor comprises the TGF-beta fusion 2 (TGFbf2) protein. Further provided herein is a composition wherein TGFbf2 comprises a human IgE signal peptide and TGF-beta variant 2 (TGFbv2). Further provided herein is a composition wherein the human IgE signal peptide comprises an amino acid sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 37. Further provided herein is a composition wherein the human IgE signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 37. Further provided herein is a composition wherein TGFbv2 comprises an amino acid sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 8. Further provided herein is a composition wherein TGFbv2 comprises the sequence set forth in SEQ ID NO: 8. Further provided herein is a composition wherein the sequence encoding a cytokine comprises a second promoter region that effects expression of the cytokine, and the sequence encoding a TGF-beta activity inhibitor comprises a third promoter region that effects expression of the TGF-beta activity inhibitor. Further provided herein is a composition wherein each of the second and third promoter regions comprises a late promoter.Further provided herein are compositions wherein the late promoter comprises any one of SSP, P7.5, P28, P135, TK promoter, E / L, F7L, H5R, H1L, A1L, J3R, E4L, I1L, I5L, I7L, T7, I2L, FP4b, ATI, P11, PFL1, PH5, L4R, 28kDa promoter, or any variation or combination thereof. Further provided herein are compositions wherein the late promoter comprises a weak late promoter. Further provided herein are compositions wherein the weak late promoter comprises a P135 promoter. Further provided herein are compositions wherein the second promoter region comprises a P135 promoter. Further provided herein are compositions wherein the late promoter comprises a strong late promoter. Further provided herein are compositions wherein the strong late promoter comprises a P7.5 promoter. Further provided herein is a composition in which the third promoter region that causes expression of a TGF-beta activity inhibitor comprises a P7.5 promoter. Further provided herein is a composition in which an exogenous nucleic acid encoding a cytokine or a functional variant thereof, an exogenous nucleic acid encoding a TGF-beta activity inhibitor, and an exogenous nucleic acid encoding a chemokine receptor or a functional variant thereof are located in a single genome. Further provided herein is a composition in which the vector is an oncolytic virus, and the oncolytic virus comprises at least one genomic modification. Further provided herein is a composition in which the at least one modification comprises a mutation or deletion of at least one gene selected from the group consisting of thymidine kinase (TK), F13L, A36R, A34R, A33R, A52R, B5R, B8R, B18R, SPI-1, SPI-2, B15R, VGF, E3L, K3L, A41L, K7R, or N1L, functional fragments thereof, or any combination thereof. Further provided herein are compositions wherein the at least one modification comprises a deletion of the genes A52R and TK.Further provided herein is a composition in which the oncolytic virus is a poxvirus, adeno-associated virus, adenovirus, reovirus, lentivirus, herpes simplex virus, vesicular stomatitis virus, mengovirus, myxoma virus, Newcastle disease virus, measles virus, or poliovirus. Further provided herein is a composition in which the poxvirus is a vaccinia virus. Further provided herein is a composition in which the vaccinia virus is a Western Reserve strain. Furthermore, at least one genome modification can increase the efficacy of tumor-targeting systemic delivery of the virus by about 2-fold, about 3-fold, about 4-fold, or about 5-fold. Provided herein are compositions that result in an increase of about 6-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 12-fold, about 14-fold, about 16-fold, about 18-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, about 55-fold, about 60-fold, about 65-fold, about 70-fold, about 75-fold, about 80-fold, about 85-fold, about 90-fold, about 95-fold, or about 100-fold.

[0158] Provided herein are nucleic acids comprising sequences encoding at least two polypeptides comprising interleukin-12 (IL-12) or a functional variant thereof and an inhibitor of transforming growth factor beta (TGF-beta) activity. Provided herein are nucleic acids comprising sequences encoding a first polypeptide comprising interleukin-12 (IL-12) or a functional variant thereof and a second polypeptide comprising an inhibitor of transforming growth factor beta (TGF-beta) activity. Also provided herein are nucleic acids comprising DNA or RNA. Also provided herein are nucleic acids in which the IL-12 is mouse IL-12 or human IL-12. Also provided herein are nucleic acids in which the IL-12 or a functional variant thereof comprises an alpha subunit and a beta subunit. Also provided herein are nucleic acids in which the alpha subunit and the beta subunit are connected by a linker. Also provided herein are nucleic acids in which the first polypeptide comprising the IL-12 alpha subunit and the IL-12 beta subunit further comprises a linker. Further provided herein are nucleic acids wherein the IL-12 alpha subunit comprises a sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 13 or SEQ ID NO: 16. Further provided herein are nucleic acids wherein the IL-12 alpha subunit comprises a sequence set forth in SEQ ID NO: 13 or SEQ ID NO: 16. Further provided herein are nucleic acids wherein the IL-12 beta subunit comprises a sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 14 or SEQ ID NO: 17. Further provided herein are nucleic acids wherein the IL-12 beta subunit comprises a sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 17. Further provided herein are nucleic acids wherein the linker comprises a sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 18. Further provided herein are nucleic acids wherein the linker comprises the sequence set forth in SEQ ID NO: 18. Further provided herein are nucleic acids wherein the encoded IL-12 comprises a sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:12 or SEQ ID NO:15.Further provided herein is a nucleic acid wherein the encoded IL-12 comprises the sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 15. Further provided herein is a nucleic acid wherein the nucleic acid encoding IL-12 comprises a sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 64 or SEQ ID NO: 66. Further provided herein is a nucleic acid wherein the nucleic acid encoding IL-12 comprises the sequence set forth in SEQ ID NO: 64 or SEQ ID NO: 66. Further provided herein is a nucleic acid wherein the TGF-beta activity inhibitor comprises a TGF-beta dominant negative, a TGF-beta receptor dominant negative, a protein that binds to TGF-beta, or a protein that binds to TGF-beta receptor. Further provided herein is a nucleic acid wherein the TGF-beta activity inhibitor comprises a protein that binds to TGF-beta, and wherein the protein that binds to TGF-beta is an antibody or a functional variant thereof. Further provided herein is a nucleic acid wherein the TGF-beta activity inhibitor comprises a protein that binds to TGF-beta receptor, and wherein the protein that binds to TGF-beta receptor is an antibody or a functional variant thereof. Further provided herein is a nucleic acid in which the TGF-beta activity inhibitor comprises a protein that binds to a TGF-beta receptor, and the protein that binds to a TGF-beta receptor is a protein comprising a TGF-beta domain. Further provided herein is a nucleic acid in which the protein comprising a TGF-beta domain comprises a sequence set forth in any one of SEQ ID NOS: 1-9. Further provided herein is a nucleic acid in which the protein comprises at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NOS: 7 or 8. Further provided herein is a nucleic acid in which the protein comprising a TGF-beta domain comprises a sequence set forth in SEQ ID NOS: 7 or 8. Further provided herein is a nucleic acid in which the nucleic acid encoding the TGF-beta activity inhibitor comprises at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NOS: 67. Further provided herein is a nucleic acid in which the nucleic acid encoding the TGF-beta activity inhibitor comprises a sequence set forth in SEQ ID NOS: 67.Further provided herein is a nucleic acid wherein the protein that binds to the TGF-beta receptor comprises a TGF-beta fusion 1 (TGFbf1) protein. Further provided herein is a nucleic acid wherein the TGFbf1 comprises a mouse IL-2 signal peptide and TGF-beta variant 1 (TGFbv1). Further provided herein is a nucleic acid wherein the mouse IL-2 signal peptide comprises a sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 36. Further provided herein is a nucleic acid wherein the mouse IL-2 signal peptide comprises the sequence set forth in SEQ ID NO: 36. Further provided herein is a nucleic acid wherein the TGFbv1 comprises a sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 7. Further provided herein is a nucleic acid wherein the TGFbv1 comprises the sequence set forth in SEQ ID NO: 7. Further provided herein is a nucleic acid wherein the protein that binds to the TGF-beta receptor comprises a TGF-beta fusion 2 (TGFbf2) protein. Further provided herein is a nucleic acid wherein TGFbf2 comprises a human IgE signal peptide and TGF-beta variant 2 (TGFbv2). Further provided herein is a nucleic acid wherein the human IgE signal peptide comprises a sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 37. Further provided herein is a nucleic acid wherein the human IgE signal peptide comprises the sequence set forth in SEQ ID NO: 37. Further provided herein is a nucleic acid wherein TGFbv2 comprises a sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 8. Further provided herein is a nucleic acid wherein TGFbv2 comprises the sequence set forth in SEQ ID NO: 8. Further provided herein is a nucleic acid wherein the protein that binds to the TGF-beta receptor binds to TGF-beta receptor II but not to TGF-beta receptor I. Further provided herein is a nucleic acid further comprising at least one promoter region. Further provided herein are nucleic acids in which at least one promoter region drives the expression of at least two polypeptides.Further provided herein is a nucleic acid in which at least one promoter region comprises a first promoter region and a second promoter region, wherein the first promoter region drives expression of a polypeptide comprising IL-12, and the second promoter region drives expression of a TGF-beta activity inhibitor. Further provided herein is a nucleic acid in which the first promoter region and the second promoter region each comprise any one of SSP, P7.5, P28, P135, TK promoter, E / L, F7L, H5R, H1L, A1L, J3R, E4L, I1L, I5L, I7L, T7, I2L, FP4b, ATI, P11, PFL1, PH5, L4R, 28kDa promoter, or any variation or combination thereof. Further provided herein is a nucleic acid in which the first promoter region comprises a P7.5 promoter. Further provided herein is a nucleic acid in which the first promoter region comprises a P135 promoter. Further provided herein is a nucleic acid wherein the second promoter region comprises a P28 promoter. Further provided herein is a nucleic acid wherein the second promoter region comprises a P7.5 promoter. Further provided herein is a nucleic acid further comprising a sequence encoding a chemokine receptor or a functional variant thereof. Further provided herein is a nucleic acid wherein the chemokine receptor comprises at least one of a CXC receptor, a CC receptor, a CX3C receptor, an XC receptor, a functional fragment thereof, a functional variant thereof, or any combination thereof. Further provided herein is a nucleic acid wherein the chemokine receptor comprises at least one of CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, CX3CR1, XCR1, a functional fragment thereof, a functional variant thereof, or any combination thereof. Further provided herein are nucleic acids wherein the chemokine receptor is CXCR3. Further provided herein are nucleic acids wherein the chemokine receptor comprises at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:42 or SEQ ID NO:43.Further provided herein is a nucleic acid in which the chemokine receptor comprises the sequence set forth in SEQ ID NO: 42 or SEQ ID NO: 43. Further provided herein is a nucleic acid in which the sequence encoding the chemokine receptor further comprises a third promoter region that causes expression of the chemokine receptor prior to expression of IL-12 and an inhibitor of TGF-beta activity. Further provided herein is a nucleic acid in which the third promoter region comprises any one of A52R, pB8, mH5, I4L, LEO, pF11, I3L, P7.5, TK promoter, F7L, H5R, short synthetic promoter (SSP), or any variation or combination thereof. Further provided herein is a nucleic acid in which the third promoter comprises an A52R promoter.

[0159] Provided herein is a nucleic acid comprising a first region encoding a first polypeptide comprising a sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:12 or SEQ ID NO:15, and a second region encoding a second polypeptide comprising a sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:40 or SEQ ID NO:41. Also provided herein is a nucleic acid comprising DNA or RNA. Also provided herein is a nucleic acid further comprising at least one promoter region. Also provided herein is a nucleic acid in which the at least one promoter region drives the expression of at least two polypeptides. Also provided herein is a nucleic acid comprising a first promoter region and a second promoter region, wherein the first promoter region drives the expression of the first polypeptide and the second promoter region drives the expression of the second polypeptide. Further provided herein are nucleic acids wherein the first and second promoter regions each comprise any one of SSP, P7.5, P28, P135, TK promoter, E / L, F7L, H5R, H1L, A1L, J3R, E4L, I1L, I5L, I7L, T7, I2L, FP4b, ATI, P11, PFL1, PH5, L4R, 28kDa promoter, or any variation or combination thereof. Further provided herein are nucleic acids wherein the first promoter region comprises the P7.5 promoter. Further provided herein are nucleic acids wherein the first promoter region comprises the P135 promoter. Further provided herein are nucleic acids wherein the second promoter region comprises the P28 promoter. Further provided herein are nucleic acids wherein the second promoter region comprises the P7.5 promoter. Further provided herein is a nucleic acid wherein a first region encodes a polypeptide comprising the sequence set forth in SEQ ID NO:12 or SEQ ID NO:15, and a second region encodes a polypeptide comprising the sequence set forth in SEQ ID NO:40 or SEQ ID NO:41.Further provided herein is a nucleic acid comprising a first region having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:64 or SEQ ID NO:66 and a second region having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:65 or SEQ ID NO:67. Further provided herein is a nucleic acid comprising DNA and comprising, in 5' to 3' order, the sequences of a first region encoding IL-12 or a functional variant thereof and a second region encoding an inhibitor of TGF-beta activity. Further provided herein is a nucleic acid comprising DNA encoding, in 5' to 3' order, the sequences set forth in SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:13, SEQ ID NO:36, and SEQ ID NO:7. Further provided herein is a nucleic acid comprising DNA encoding, in 5' to 3' order, the sequences encoding SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:16, SEQ ID NO:37, and SEQ ID NO:8. Further provided herein is a nucleic acid comprising, in 5' to 3' order, the sequences encoding SEQ ID NO:12 and SEQ ID NO:40. Further provided herein is a nucleic acid comprising, in 5' to 3' order, a sequence encoding SEQ ID NO: 15 and SEQ ID NO: 41. Further provided herein is a nucleic acid further comprising a third region encoding a third polypeptide comprising a sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 42 or SEQ ID NO: 43. Further provided herein is a nucleic acid wherein the third region comprises SEQ ID NO: 42 or SEQ ID NO: 43. Further provided herein is a nucleic acid further comprising a third region having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 69 or SEQ ID NO: 86. Further provided herein is a nucleic acid wherein the third region comprises the sequence set forth in SEQ ID NO: 69 or SEQ ID NO: 86. Further provided herein is a nucleic acid wherein the third region encoding the third polypeptide further comprises a third promoter region that effects expression of the third polypeptide prior to expression of the first and second polypeptides.Further provided herein is a nucleic acid wherein the third promoter comprises any one of A52R, pB8, mH5, I4L, LEO, pF11, I3L, P7.5, TK promoter, F7L, H5R, short synthetic promoter (SSP), or any variation or combination thereof. Further provided herein is a nucleic acid wherein the third promoter comprises an A52R promoter. Further provided herein is a nucleic acid present in an oncolytic virus. Further provided herein is a nucleic acid wherein the oncolytic virus is a poxvirus, adeno-associated virus, adenovirus, reovirus, lentivirus, herpes simplex virus, vesicular stomatitis virus, mengovirus, myxoma virus, Newcastle disease virus, measles virus, or poliovirus. Further provided herein is a nucleic acid wherein the poxvirus is a vaccinia virus. Further provided herein are nucleic acids wherein the vaccinia virus is Western Reserve vaccinia virus (ATCC VR-1354), vaccinia virus Ankara (ATCC VR-1508), vaccinia virus Ankara (ATCC VR-1566), vaccinia virus strain Wyeth (ATCC VR-1536), or a modified strain of vaccinia virus Wyeth (ATCC VR-325). Further provided herein are nucleic acids that are inserted into a viral genome. Further provided herein are nucleic acids that further comprise a mutation or deletion in at least one viral gene selected from the group consisting of thymidine kinase (TK), F13L, A36R, A34R, A33R, A52R, B5R, B8R, B18R, SPI-1, SPI-2, B15R, VGF, E3L, K3L, A41L, K7R, or N1L, a functional fragment thereof, or any combination thereof.

[0160]

[0003] Provided herein is a nucleic acid molecule comprising, in 5' to 3' order, a first promoter region whose promoter comprises the A52R promoter, an A52R locus comprising an insertion of a first region encoding human CXCR3, and an insertion at the TK locus comprising, in 5' to 3' order, a second promoter region whose promoter comprises P135, a second region encoding human IL-12, a third promoter region whose promoter comprises P7.5, and a third region encoding a TGF-beta variant.

[0004] Further provided herein is a nucleic acid molecule in which the P135 promoter comprises the sequence set forth in SEQ ID NO: 56 and the P7.5 promoter comprises the sequence set forth in SEQ ID NO: 57.

[0005] Further provided herein is a nucleic acid molecule in which the first region encoding human CXCR3 comprises the sequence set forth in SEQ ID NO: 86, the second region encoding human IL-12 comprises the sequence set forth in SEQ ID NO: 66, and the third region encoding a TGF-beta inhibitor comprises the sequence set forth in SEQ ID NO: 67. Further provided herein are nucleic acid molecules comprising an insertion in the A52R locus comprising the sequence set forth in SEQ ID NO:88, and an insertion in the TK locus comprising the sequence set forth in SEQ ID NO:85.

[0161] Provided herein is a pharmaceutical composition comprising a nucleic acid described herein or a vector described herein and a pharmaceutically acceptable excipient. Also provided herein is a pharmaceutical composition in which the composition is in a liquid dosage form. Also provided herein is a pharmaceutical composition in which the pharmaceutically acceptable excipient is a buffered saline solution. Also provided herein is a pharmaceutical composition in which the buffered saline solution is phosphate-buffered saline (PBS), Dulbecco's PBS (DPBS), TRIS-buffered saline (TBS), Hank's balanced salt solution (HBSS), Earl's balanced salt solution (EBSS), normal citrate saline (SSC), HEPES-buffered saline (HBS), or Gey's balanced salt solution. Also provided herein is a pharmaceutical composition in which the composition further comprises liposomes or nanoparticles. Also provided herein is a pharmaceutical composition in which the nucleic acid or vector is associated with liposomes or nanoparticles.

[0162] Provided herein are methods for treating cancer, comprising administering to a subject with cancer a pharmaceutical composition described herein in an amount sufficient to treat the cancer. Further provided herein are methods wherein the cancer is a solid tumor or a blood cancer. Further provided herein are methods wherein the cancer includes melanoma, hepatocellular carcinoma, breast cancer, lung cancer, peritoneal cancer, prostate cancer, bladder cancer, ovarian cancer, leukemia, lymphoma, kidney cancer, pancreatic cancer, epithelial cancer, gastric cancer, colon cancer, duodenal cancer, pancreatic adenocarcinoma, mesothelioma, glioblastoma multiforme, astrocytoma, multiple myeloma, prostate cancer, hepatocellular carcinoma, cholangiosarcoma, pancreatic adenocarcinoma, head and neck squamous cell carcinoma, colorectal cancer, gastrointestinal-type gastric adenocarcinoma, cervical squamous cell carcinoma, osteosarcoma, epithelial ovarian cancer, acute lymphoblastic lymphoma, myeloproliferative neoplasm, or sarcoma. Further provided herein are methods wherein the administering step comprises intratumoral administration. Further provided herein are methods wherein the administering step comprises systemic administration. Further provided herein are methods wherein the systemic administration comprises oral administration, parenteral administration, intranasal administration, sublingual administration, rectal administration, transdermal administration, or any combination thereof.

[0163] Provided herein is a method for activating an anti-tumor immune response, comprising administering a pharmaceutical composition described herein to a subject with cancer. Further provided herein is a method wherein the cancer is a solid tumor, leukemia, or lymphoma. Further provided herein is a method wherein the cancer includes melanoma, hepatocellular carcinoma, breast cancer, lung cancer, peritoneal cancer, prostate cancer, bladder cancer, ovarian cancer, leukemia, lymphoma, kidney cancer, pancreatic cancer, epithelial cancer, gastric cancer, colon cancer, duodenal cancer, pancreatic adenocarcinoma, mesothelioma, glioblastoma multiforme, astrocytoma, multiple myeloma, prostate cancer, hepatocellular carcinoma, cholangiosarcoma, pancreatic adenocarcinoma, head and neck squamous cell carcinoma, colorectal cancer, gastrointestinal-type gastric adenocarcinoma, cervical squamous cell carcinoma, osteosarcoma, epithelial ovarian cancer, acute lymphoblastic lymphoma, myeloproliferative neoplasm, or sarcoma. Further provided herein is a method wherein the administering step is intratumoral administration. Further provided herein are methods wherein the administering step is systemic administration. Further provided herein are methods wherein the systemic administration includes oral administration, parenteral administration, intranasal administration, sublingual administration, rectal administration, transdermal administration, or any combination thereof.

[0164]

[0003] Provided herein is a method for reducing the incidence of tumor cell growth, comprising administering to tumor cells a pharmaceutical composition described herein in an effective amount sufficient to reduce the incidence of tumor cell growth. Further provided herein is a method wherein the tumor cells are derived from a solid tumor or a blood cancer. Further provided herein is a method wherein the tumor cells are derived from melanoma, hepatocellular carcinoma, breast cancer, lung cancer, peritoneal cancer, prostate cancer, bladder cancer, ovarian cancer, leukemia, lymphoma, kidney cancer, pancreatic cancer, epithelial cancer, gastric cancer, colon cancer, duodenal cancer, pancreatic adenocarcinoma, mesothelioma, glioblastoma multiforme, astrocytoma, multiple myeloma, prostate cancer, hepatocellular carcinoma, cholangiosarcoma, pancreatic adenocarcinoma, head and neck squamous cell carcinoma, colorectal cancer, gastrointestinal-type gastric adenocarcinoma, cervical squamous cell carcinoma, osteosarcoma, epithelial ovarian cancer, acute lymphoblastic lymphoma, myeloproliferative neoplasm, or sarcoma. Further provided herein are methods wherein the administering step is intratumoral administration. [Example]

[0165] The following examples further illustrate the described embodiments without limiting the scope of the disclosure.

[0166] Example 1 Design of mIL12 and TGFbf1 constructs Vaccinia viruses containing the constructs shown in Figure 2 were constructed by replacing the open reading frame encoding the viral thymidine kinase with nucleic acids containing sequences encoding murine IL-12 (murine IL-12) (SEQ ID NO: 64) and TGF-beta fusion (TGF-bf1) (SEQ ID NO: 65), as shown in Table 9. Expression of murine IL-12 was driven by viral promoter P7.5 (SEQ ID NO: 57). Expression of TGF-bf1 was driven by viral promoter P28 (SEQ ID NO: 58). The murine IL-12 polypeptide comprised a covalent dimer consisting of murine interleukin-12 subunit beta (mIL-12b) (UniProtKB Accession ID P43432.I) (SEQ ID NO: 14) linked to murine interleukin-12 subunit alpha (mIL-12a) (UniProtKB Accession ID 43431) residues 11-215 (SEQ ID NO: 13) by a 22-residue glycine-rich linker (SEQ ID NO: 18). The TGFbf1 polypeptide contained the signal peptide of mouse interleukin-2 (IL-2sig) (UniProtKB Accession ID P04351.1) (SEQ ID NO: 36) fused to TGF-beta inhibitor (TGFbv1) (SEQ ID NO: 7) in which cysteines 8 and 17 (original PDB numbering) were mutated to valine and alanine, respectively. Table 9. Nucleic acid sequences. [Table 9]

[0167] Example 2 Design of hIL12 and TGFbf2 constructs Vaccinia viruses containing the constructs shown in Figure 2 were constructed by replacing the open reading frame encoding the viral thymidine kinase with nucleic acids containing sequences encoding human IL-12 (SEQ ID NO: 66) and TGF-beta fusion (TGF-bf1) (SEQ ID NO: 67), as shown in Table 10. Expression of hIL-12 was driven by viral promoter P135 (SEQ ID NO: 57). Expression of TGF-bf2 was driven by viral promoter P7.5 (SEQ ID NO: 57). The human IL-12 polypeptide comprises a covalent dimer consisting of human interleukin-12 subunit beta (hIL-12b) (SEQ ID NO: 17) linked to human interleukin-12 subunit alpha (hIL-12a) (SEQ ID NO: 16) by a 22-residue glycine-rich linker (SEQ ID NO: 18). The TGFbf2 polypeptide comprises the signal peptide of human IgE (SEQ ID NO: 37) fused to a TGF-beta inhibitor containing the cystine-knot region of PRDC (TGFbv2) (SEQ ID NO: 8). Table 10. Nucleic acid sequences. [Table 10]

[0168] Example 3 Measurement of tumor growth after treatment with TGF-beta inhibitors and IL-12 Renca cells were implanted into the flanks of Balb / c mice. After 12 days, mice were transplanted into each treatment group with an average of 58 mm 3 Mice were treated with 1E7 PFU of virus by IT injection.

[0169] B16F10 cells were mixed 1:1 with Matrigel and implanted into the flanks of C57 / Black 6 mice. After 5 days, mice were implanted into the flanks of each treatment group, with an average of 93 mm 3 Mice were treated with 1E7 PFU of virus by IT injection.

[0170] Tumors were measured twice weekly and mice were weighed weekly. Mice were monitored for signs of morbidity and mortality, none of which were observed (data not shown).

[0171] Tumor-bearing mice were divided into treatment groups, including PBS, TK-, IL12-expressing TK-, TGF-beta inhibitor-expressing TK-, and IL-12 and TGF-beta inhibitor-expressing TK-. The mean tumor volume was measured in each treatment group, as shown in Figure 3A for the Renca tumor group and Figure 3B for the B16 tumor group.

[0172] Renca tumor-inducing mice treated with a virus expressing a TGF-beta inhibitor alone grew to approximately 225 mm after 17 days of treatment. 3 The groups treated with viruses expressing a combination of IL-12 and TGF-beta inhibitors or IL-12 alone showed a mean tumor volume of 50 mm 17 days after treatment. 3 In comparison, control mice treated with PBS had a mean tumor volume of approximately 450 mm 3 The mean tumor volume was 1.01.

[0173] B16 tumor-induced mice treated with a virus expressing a TGF-beta inhibitor alone grew tumors of approximately 600 mm after 17 days of treatment. 3 Mice treated with viruses expressing a combination of IL-12 and TGF-beta inhibitors or IL-12 alone had a mean tumor volume of 100 mm 17 days after treatment. 3 In comparison, control mice treated with PBS had a mean tumor volume of approximately 1200 mm 3 The mean tumor volume was 1.01.

[0174] Treatment with the modified virus expressing both a TGF-beta inhibitor and IL-12 inhibited tumor growth in mice more than treatment with a virus expressing the TGF-beta inhibitor alone.

[0175] Example 4 Survival assay with TGF-beta inhibitor and mIL-12 Tumors were induced in mice using Renca and B16 cells and then treated with the modified virus described in Example 2. Treatment groups included PBS, TK-virus, TK-virus expressing IL12, TK-virus expressing a TGF-beta inhibitor, and TK-virus expressing IL-12 and a TGF-beta inhibitor. Survival probability was calculated for each treatment group, as shown in Figure 4A for the Renca tumor group and in Figure 4B for the B16 tumor group. In mice bearing Renca cell tumors, treatment groups receiving viruses expressing IL-12 or IL-12 and a TGF-beta inhibitor showed approximately 75% survival after 56 days, while no subjects in the other groups survived by day 56. In mice bearing B16 tumors, those treated with the modified virus expressing a TGF-beta inhibitor and IL-12 showed approximately 35% survival, compared with no subjects in the other treatment groups.

[0176] Example 5 Addition of mouse CXCR3 expression system Vaccinia virus was modified by replacing the gene encoding A52 with nucleic acids encoding mouse CXCR3 and a fluorescent reporter. A plasmid transfer vector was generated containing, without a gap, the upstream recombination-inducing sequence A (SEQ ID NO: 68), an open reading frame encoding mouse CXCR3 (SEQ ID NO: 69), a stop codon, a SacI cloning site and a short spacer (SEQ ID NO: 70), a loxP site (SEQ ID NO: 71), a viral promoter driving expression of the GFP-pac reporter (SEQ ID NO: 72), a PacI cloning site and a short spacer A (SEQ ID NO: 73), a loxP site (SEQ ID NO: 71), and a downstream recombination-inducing sequence A (SEQ ID NO: 74). After recombination and treatment with cre recombinase, the viral genome contained the integrated sequence set forth in SEQ ID NO: 75. The sequence is shown in Table 11. A schematic diagram of the promoter and gene expression construct is shown in Figure 5. Table 11. Mouse CXCR3 recombinant sequence. [Table 11-1] [Table 11-2] [Table 11-3] [Table 11-4]

[0177] Example 6 Construction of mouse IL-12 and TGF-beta inhibitor expression systems The modified vaccinia virus of Example 5 was further modified by replacing the gene encoding thymidine kinase (VACV094, J2R) with nucleic acids encoding single-chain murine IL-12, TGF-bf2, and a fluorescent reporter. A plasmid transfer vector was generated containing, without gaps, the following sequence: upstream recombination inducer sequence B (SEQ ID NO: 76), an SbfI cloning site followed by the P135 promoter (SEQ ID NO: 77), an open reading frame encoding murine IL-12 (SEQ ID NO: 64), a SalI cloning site followed by a spacer (SEQ ID NO: 78), the vaccinia virus promoter P7.5 followed by a KpnI cloning site (SEQ ID NO: 79), an open reading frame encoding a TGF-beta inhibitor (TGFbf2) (SEQ ID NO: 67), a sacI cloning site (SEQ ID NO: 81), a loxP site (SEQ ID NO: 71), a spacer followed by the viral promoter driving expression of the GFP-pac reporter (SEQ ID NO: 72), a PacI cloning site and a short spacer B (SEQ ID NO: 82), a loxP site (SEQ ID NO: 71), and a downstream recombination inducer sequence B (SEQ ID NO: 83). After recombination and treatment with cre recombinase, the viral genome incorporated the sequence set forth in SEQ ID NO: 84. The selected sequences are shown in Table 12. A schematic diagram of the promoter and gene expression construct is shown in Figure 6. Table 12. Mouse IL-12 and TGF beta inhibitor recombinant sequences. [Table 12-1] [Table 12-2] [Table 12-3] [Table 12-4] [Table 12-5]

[0178] Example 7 Construction of human IL-12 and TGF-beta inhibitor expression systems Vaccinia virus was modified by replacing the gene encoding thymidine kinase (VACV094, J2R) with nucleic acids encoding single-chain human IL-12, the TGF-β inhibitor TGFbf2, and a fluorescent reporter. A plasmid transfer vector was generated containing, without gaps, the following sequence: upstream recombination inducer sequence B (SEQ ID NO: 76), an SbfI cloning site followed by the P135 promoter (SEQ ID NO: 77), an open reading frame encoding human IL-12 (SEQ ID NO: 66), a SalI cloning site followed by a spacer (SEQ ID NO: 78), the vaccinia virus promoter P7.5 followed by a KpnI cloning site (SEQ ID NO: 79), an open reading frame encoding TGF-beta inhibitor (TGFbf2) (SEQ ID NO: 67), a sacI cloning site (SEQ ID NO: 81), a loxP site (SEQ ID NO: 71), a spacer followed by the viral promoter driving expression of the GFP-pac reporter (SEQ ID NO: 72), a PacI cloning site and a short spacer B (SEQ ID NO: 82), a loxP site (SEQ ID NO: 71), and a downstream recombination inducer sequence B (SEQ ID NO: 83). After recombination and treatment with cre recombinase, the viral genome contained the integrated sequence set forth in SEQ ID NO: 85. The sequence is shown in Table 13. Table 13. Human IL-12 and TGF beta inhibitor recombinant sequences. [Table 13-1] [Table 13-2] [Table 13-3] [Table 13-4]

[0179] Example 8 Addition of chemokine receptor sequences The modified vaccinia virus described in Example 7 was further modified by replacing the gene encoding A52 (VACV178, A52R) with nucleic acids encoding human CXCR3 and a fluorescent reporter. A plasmid transfer vector was generated containing, in order without a gap, an upstream recombination sequence A (SEQ ID NO: 68), an open reading frame encoding human CXCR3 isoform 1 (SEQ ID NO: 86), a stop codon, a SacI cloning site and a short spacer (SEQ ID NO: 70), a loxP site (SEQ ID NO: 71), the spacer followed by a viral promoter driving expression of the GFP-pac reporter (SEQ ID NO: 72), a PacI cloning site and a short spacer A (SEQ ID NO: 73), a loxP site (SEQ ID NO: 71), and a downstream recombination sequence A (SEQ ID NO: 74). Reporter-positive viruses were isolated and then treated with a reporter-free transfer vector containing an upstream recombinogenic sequence A (SEQ ID NO: 68), an open reading frame encoding human CXCR3 isoform 1 (SEQ ID NO: 86), and a second downstream recombinogenic sequence (SEQ ID NO: 87). After recombination, the viral genome incorporated the sequence set forth in SEQ ID NO: 88. Selected sequences are shown in Table 14. Table 14. Human CXCR3 recombinant sequences. [Table 14-1] [Table 14-2] [Table 14-3] [Table 14-4]

[0180] Example 9 Generation of modified oncolytic vaccinia viruses expressing CXCR3, IL-12, and TGFB inhibitors As shown in Figure 7, modified oncolytic vaccinia viruses expressing CXCR3 described by SEQ ID NO: 43, IL-12 described by SEQ ID NO: 15, and TGFB1 antagonistic minimonomer constructs described by SEQ ID NO: 41 were generated according to the methods described in Examples 7 and 8. Expression and function of the transgenes in the modified viruses were confirmed as follows.

[0181] CXCR3 Fluorescence-activated cell sorting (FACS) analysis of HeLa cells infected with the modified viruses, shown in Figure 8A, confirms CXCR3 expression. The function of expressed CXCR3 was examined in peripheral blood mononuclear cells (PBMCs), including CD4+ cells, CD8+ cells, monocytes, and B cells. Subpopulations of PBMCs infected with the modified viruses were compared to control (control) or uninfected (-) cells infected with TK-virus for migration toward 100 ng / ml of ITAC (CXCL11), a CXCR3 ligand. The counts of migrating cells in each test group are shown as bar plots in Figures 8B-8E. Figure 8B shows a greater than four-fold increase in migration of CD4 cells infected with the modified viruses compared to control cells. Figure 8C shows an approximately 2.5-fold increase in migration of CD8 cells infected with the modified viruses compared to control cells. Figure 8D shows monocyte migration toward infected cells compared to no response in control cells. Figure 8E shows an approximately 10-fold increase in migration of B cells infected with the modified virus compared to control cells. The increased migration toward CXCR3 ligand indicates expression of the active form of the receptor in cells infected with the modified virus.

[0182] IL-12 ELISA assays of culture supernatants from HeLa cells infected with the modified viruses confirmed the expression of IL-12 bearing the conserved epitope. Figure 8F is a bar plot showing the results of quantitative ELISA obtained from the supernatants of HeLa cells infected with the modified viruses compared with controls infected with TK-virus (control) or uninfected cells (-). Approximately 170,000 pg / ml of IL-12 was detected in the supernatants obtained from cells infected with the modified viruses. IL-12 was not detected in untreated or negative control cells.

[0183] TGFBi HeLa cell culture lysates obtained from cells infected with the modified virus, a control infected with TK-virus (control), or uninfected cells (-) were analyzed using Western blot. As shown in Figure 8G, lysates obtained from cells infected with the modified virus exhibited a 12 kDa protein corresponding to the TGFBi protein. Untreated and negative control cultures did not exhibit a corresponding band.

[0184] Example 10 The engineered virus rescues CD8 T cells from TGF-beta suppression and induces increased granzyme B production HeLa cells were treated with medium (-) or infected with TK-control virus (control) or modified virus (TGFBi / IL-12 / CXCR3) at a multiplicity of infection (MOI) of 10 for 24 hours. Virus-free supernatants were collected from infected cells and used for subsequent T cell experiments. CD8+ T cells treated with supernatants obtained from HeLa cells infected with the modified virus exhibited less suppression upon treatment with TGF-beta 1 (TGFB1), indicating the expression of a functional TGFB inhibitor. CD8+ T cells were labeled with 2 mM CFSE and stimulated with anti-CD3 and anti-CD28 antibodies in the presence of supernatants obtained from HeLa cells infected with the modified virus or TK-control virus (control) or uninfected cells. T cells were then exposed to 0 ng / ml, 10 ng / ml, or 50 ng / ml TGFB1.

[0185] T cells were analyzed for CD44 and GZMB expression. T cells were stained with antibodies for surface-expressed CD44 and then fixed and permeabilized using a Cytofix / Cytoperm™ fixation / permeabilization kit (BD Biosciences, Franklin Lakes, NJ). Cells were stained with antibodies for cytoplasmic GZMB and analyzed using an Attune flow cytometer (Thermo Fisher Scientific, Carlsbad, CA).

[0186] The initial population of cells that incorporated the dye appears as the peak with the highest intensity in the trace. Subsequent generations of cells contain diluting amounts of dye and appear as diminishing fluorescence or peaks progressing to the left. Cells sensitive to TGFB1 inhibition show smaller generation peaks in the trace. The trace shown in Figure 9A shows a decrease in generation peaks in TK-control virus-infected (control) and uninfected (-) CD8 T cells after exposure to 10 ng / ml and 50 ng / ml of TGFB1, indicating inhibition of proliferation. T cells treated and stimulated with the modified virus showed less inhibition of proliferation after contact with TGFB1 compared to controls.

[0187] CD8 cells were also assessed for granzyme B (GZMB) induction. Attune-generated FCS files were analyzed using FCS 7 software. Gating was performed on viable T cells. Density plots were generated with CD44 on the X-axis and GZMB on the Y-axis. The percentage of GZMB was analyzed by gating on GZMB+ T cells in the density plot. Figure 9B shows dot plots for each sample group. The plots demonstrate increased levels of CD44+ GZMB+ in CD8 T cells infected with the modified viruses compared to untreated (-) and TK-virus-infected control cells.

[0188] Example 11 In vitro selectivity and enhanced in vivo tumor-specific delivery of engineered vaccinia viruses The selectivity of the modified virus was tested compared to the control virus using an in vivo mouse RENCA tumor model. The modified virus described in Example 9 was tested for infectivity in cancer and non-cancerous cell lines. Three cell types, human lung adenocarcinoma (A549) cells, human cervical carcinoma (Hela) cells, and human foreskin fibroblasts (HFF), were analyzed for virus load 48 hours after infection. The detected PFU / ml in each cell type is shown in the bar graph in Figure 10A. 48 hours after infection, Hela cells detected 10 times more PFU / ml of virus than HFF cells. A549 cells showed approximately 4 times higher virus load than infected HFF cells. The modified virus exhibits preferential infectivity in tumor cells in vitro.

[0189] Renca tumors were implanted subcutaneously in BALB / c mice. 7 PFU of the modified virus was intravenously administered to mice with and without the CXCR3-expressing modification. Tumors were harvested 24 hours later, and viral genomes were quantified by qPCR. The results are shown in the scatter plot in Figure 10B. Approximately 10 times more viral genomes were detected in Renca tumors in mice treated with the CXCR3-expressing virus than in tumors obtained from mice treated with the virus without CXCR3 modification. The results showed that the modified virus had increased in vivo infectivity compared to the unmodified virus.

[0190] Example 12 Modified vaccinia virus reduces tumor burden and increases mouse survival in multiple tumor models The therapeutic efficacy of the modified viruses was tested in vivo in mouse EMT-6 and MC38 tumor models compared to control virus and buffer negative controls. Mice were inoculated with EMT6 and MC38 tumors.

[0191] BalbC mice, 1 x 10 5 10 EMT6 cells were implanted. Thirteen days after implantation, the tumors grew to a size of 80.26 mm 3 The average volume reached (26.70-136.23 mm 3 ). 36.92mm 3 and mice bearing tumors smaller than 117.43 mm 3 Mice with tumors larger than 78 mm were excluded, and mice were divided into groups (n=10) with tumors of approximately 78 mm 3 Mice were grouped into groups with a mean tumor volume of 1 x 10 4 , 1×10 5 , 1×10 6 , or 1 × 10 7 Mice were treated with PFU of vaccinia virus (TGFBi / IL-12 / CXCR3) modified as described in Example 9, buffer (-), or TK-control virus (control). Treatment was administered by IV injection (tail vein) on days 0 and 3. Tumor volume was measured twice weekly, and mice were weighed weekly.

[0192] C57Bl / 6 mice were treated with 5 × 10 5 The cells were implanted and allowed to form tumors over a period of eight days, growing to a size of 50.46 mm. 3 The average was 10.17-84.79 mm. 3 ). 31.99mm 3 Mice with tumors smaller than 53.06 mm were excluded, and mice were divided into groups (n=10) with tumors smaller than 53.06 mm. 3 Mice were divided into groups with an average volume of 1 x 10 5 , 2 times 1 × 10 6 Mice were treated with a single 1×10 PFU / dose of vaccinia virus (TGFBi / IL-12 / CXCR3) modified as described in Example 9, buffer (−), or TK-control virus (control). Treatment was by IV injection (tail vein) on days 0 and 3. Tumors were measured twice weekly, and mice were weighed weekly.

[0193] All buffer control (-) and control virus (control)-treated mice in both tumor models were euthanized by day 42. Eight of ten EMT6 mice treated with the modified virus showed complete suppression of tumor growth by day 54. MC38 mice treated with the modified virus showed complete suppression of tumor growth for more than 39 days, with nine of ten mice showing complete suppression by day 51. The survival probability of EMT6 and MC38 tumor mice up to 54 days after treatment is shown in Figures 11C and 11D, respectively. Asterisks indicate P values: *P≦0.05, **P≦0.01, ***P≦0.001, and ****P≦0.0001. As shown in Figure 11C, mice treated with the modified virus maintained a 70% survival probability by day 54. As shown in Figure 11D, mice treated with the modified virus maintained a 90% survival probability by day 54. The results show that treatment with the modified virus suppressed tumor growth and improved survival in multiple tumor models.

[0194] Example 13 Treatment with the modified virus increases CD3+CD8+ T cell infiltration in tumors Postmortem analysis was used to analyze the effects of the modified viruses on the tumor immune / stromal / endothelial environment and determine the toxicity profile. Renca tumors from Example 11 and MC38 tumors from Example 12 were sectioned and processed for nuclear, CD3, and CD8 staining. Representative stained sections are shown in Figure 12A. Tumors treated with the modified viruses showed greater infiltration of CD3 and CD8 markers, indicating infiltration of cytotoxic T cells, compared to corresponding sections obtained from untreated or control samples.

[0195] Figures 12B-12E are bar plots of the total counts of CD3+ and CD8+ T cells in RENCA and MC38 tumor samples after treatment with the modified viruses or untreated or control samples. Figure 12B shows an approximately 3-fold increase in CD3+ T cells in RENCA tumors treated with the modified viruses compared to control cells. Figure 12C shows an approximately 3.5-fold increase in CD8+ T cells in RENCA tumors treated with the modified viruses compared to control cells. Figure 12D shows an approximately 2-fold increase in the average number of CD3+ T cells after treatment with the modified viruses compared to control cells. Figure 12E shows an approximately 2-3-fold increase in the average number of CD8+ T cells after treatment with the modified viruses compared to control cells.

[0196] Example 14 The engineered virus induces a type II interferon-G-associated gene signature but abolishes a TGFB1-associated gene signature in tumors Whole RENCA tumors were harvested from treated mice as described in Example 11 and homogenized in Qiazol (Qiagen, Hilden, Germany) using a bead mill. RNA was extracted using the Rneasy kit (Qiagen). The extracted RNA was sequenced using next-generation sequencing methods. Raw RNA-seq data (FASTQ) files were analyzed for differential gene expression between the TK-control virus (control) and the modified virus using Rosalind software, and gene expression heat maps were generated, as shown in Figures 13A and 13B.

[0197] Figure 13A is a heat map showing the relative expression levels of type II interferon-gamma (IFNG)-related genes, particularly compared to the overall average. Expression levels were tested for CXCL11, XCR1, STAT1, IDO1, IL12B, IFNG, CIITA, H2-EB1, H2-AB1, TBX21, CXCR3, CD2, LTB, CXCL16, B2M, VCAM1, TAP1, IFIT2, TAP2, IL2RG, STAT2, CD274, and IRF1. Area 1 shows that control CD3+ and CD8+ cells generally exhibit low expression levels compared to the overall average, while area 2 shows that CD3+ and CD8+ cells treated with modified viruses exhibit high expression levels. This indicates that contacting cells with modified viruses expressing TGF-beta inhibitors activates the expression of IFNG-related genes.

[0198] Figure 13B is a heat map showing the relative expression levels of TGF-beta 1-related genes compared to the overall average. Expression levels were tested for ILz1B, LPL, SLP1, FBN1, LCN2, CXCL5, OGN, PLOD2, TNFAIP6, CAN, ABCG1, ACKR3, and COL15A1. Area 3 shows that control cells generally exhibit high expression levels compared to the overall average, while area 4 shows that cells treated with modified viruses exhibit low expression levels. This indicates that contacting cells with modified viruses expressing TGF-beta inhibitors reduces the expression of TGF-beta 1-related genes.

[0199] The foregoing description and accompanying drawings describe certain presently representative embodiments. Various modifications, additions, and alternative designs will, of course, be apparent to those skilled in the art in light of the foregoing teachings without departing from the scope thereof, which is indicated not by the foregoing description but by the following claims. All changes and variations that come within the meaning and range of equivalency of the claims are to be embraced within their scope.

Claims

1. A composition comprising a vector, the vector comprising: an exogenous nucleic acid comprising a sequence encoding a cytokine or a functional variant thereof; an exogenous nucleic acid comprising a sequence encoding a chemokine receptor or a functional variant thereof; a first promoter region upstream to the sequence encoding said chemokine receptor, causing expression of said chemokine receptor prior to expression of said cytokine; A composition comprising:

2. 2. The composition of claim 1, wherein the encoded chemokine receptor comprises at least one of a CXC receptor, a CC receptor, a CX3C receptor, an XC receptor, a functional fragment thereof, a functional variant thereof, or any combination thereof.

3. 2. The composition of claim 1, wherein the encoded chemokine receptor comprises at least one of CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, CX3CR1, XCR1, a functional fragment thereof, or a functional variant thereof, or any combination thereof.

4. The composition of claim 1 , wherein the encoded chemokine receptor comprises CXCR3.

5. The composition of claim 4, wherein the encoded chemokine receptor comprises an amino acid sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:42 or SEQ ID NO:

43.

6. The composition of claim 4, wherein the encoded chemokine receptor comprises the amino acid sequence set forth in SEQ ID NO: 42 or SEQ ID NO:

43.

7. The composition of claim 1 , wherein the first promoter region comprises an early promoter.

8. 8. The composition of claim 7, wherein the early promoter comprises any one of A52R, pB8, mH5, I4L, LEO, pF11, I3L, P7.5, TK promoter, F7L, H5R, a short synthetic promoter (SSP), or any variation or combination thereof.

9. The composition of claim 7 , wherein the early promoter comprises an A52R promoter.

10. The composition of claim 1, wherein the encoded cytokine comprises IL-12 or a functional variant thereof.

11. The composition of claim 10, wherein the encoded IL-12 is mouse IL-12 or human IL-12.

12. The composition of claim 10, wherein the encoded IL-12 comprises an alpha subunit and a beta subunit.

13. 13. The composition of claim 12, wherein the sequences encoding the IL-12 alpha subunit and the IL-12 beta subunit further comprise a sequence encoding a linker.

14. 13. The composition of claim 12, wherein the encoded IL-12 alpha subunit comprises an amino acid sequence having at least 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NO:13 or SEQ ID NO:

16.

15. 13. The composition of claim 12, wherein the encoded IL-12 alpha subunit comprises the amino acid sequence set forth in any one of SEQ ID NO:13 or SEQ ID NO:

16.

16. 13. The composition of claim 12, wherein the encoded IL-12 beta subunit comprises an amino acid sequence having at least 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NO:14 or SEQ ID NO:

17.

17. 13. The composition of claim 12, wherein the IL-12 beta subunit comprises the amino acid sequence set forth in any one of SEQ ID NO: 14 or SEQ ID NO:

17.

18. The composition of claim 13 , wherein the encoded linker comprises an amino acid sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

18.

19. The composition of claim 13 , wherein the encoded linker comprises the amino acid sequence set forth in SEQ ID NO:

18.

20. 11. The composition of claim 10, wherein the encoded IL-12 comprises an amino acid sequence having at least 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NO:12 or SEQ ID NO:

15.

21. 11. The composition of claim 10, wherein the encoded IL-12 comprises the amino acid sequence set forth in any one of SEQ ID NO:12 or SEQ ID NO:

15.

22. 11. The composition of claim 10, wherein the sequence encoding IL-12 comprises a nucleic acid sequence having at least 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NO:64 or SEQ ID NO:

66.

23. 11. The composition of claim 10, wherein the sequence encoding IL-12 comprises the nucleic acid sequence set forth in any one of SEQ ID NO:64 or SEQ ID NO:

66.

24. further comprising an exogenous nucleic acid comprising a sequence encoding an inhibitor of transforming growth factor beta (TGF-beta) activity; The composition of any one of claims 1 to 23, wherein the first promoter region causes expression of the chemokine receptor before expression of the inhibitor of TGF-beta activity.

25. 25. The composition of claim 24, wherein the encoded inhibitor of TGF-beta activity comprises a TGF-beta dominant negative, a TGF-beta receptor dominant negative, a protein that binds to TGF-beta, or a protein that binds to a TGF-beta receptor.

26. The composition of claim 25, wherein the encoded protein that binds to the TGF-beta receptor is a protein containing a TGF-beta domain.

27. The composition of claim 26, wherein the encoded protein comprising the TGF-beta domain comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 9.

28. 27. The composition of claim 26, wherein the encoded protein comprising a domain of TGF-beta comprises an amino acid sequence having at least 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NO: 7 or SEQ ID NO:

8.

29. 27. The composition of claim 26, wherein the encoded protein comprising the TGF-beta domain comprises the amino acid sequence set forth in any one of SEQ ID NO: 7 or SEQ ID NO:

8.

30. 25. The composition of claim 24, wherein the sequence encoding the inhibitor of transforming growth factor beta (TGF-beta) activity comprises a nucleic acid sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

67.

31. The composition of claim 24, wherein the sequence encoding the inhibitor of TGF-beta activity comprises the nucleic acid sequence set forth in SEQ ID NO:

67.

32. 26. The composition of claim 25, wherein the encoded protein that binds to the TGF-beta receptor is a TGF-beta fusion protein.

33. The composition of claim 25, wherein the encoded inhibitor of TGF-beta activity comprises a protein that binds to the TGF-beta receptor, and the protein that binds to the TGF-beta receptor comprises TGF-beta fusion 1 (TGFbf1) protein.

34. 34. The composition of claim 33, wherein the TGFbf1 protein comprises a mouse IL-2 signal peptide and TGF-beta variant 1 (TGFbv1).

35. 35. The composition of claim 34, wherein the mouse IL-2 signal peptide comprises an amino acid sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

36.

36. 35. The composition of claim 34, wherein the mouse IL-2 signal peptide comprises the amino acid sequence set forth in SEQ ID NO:

36.

37. 35. The composition of claim 34, wherein the TGFbvl comprises an amino acid sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

7.

38. 35. The composition of claim 34, wherein the TGFbv1 comprises the sequence set forth in SEQ ID NO:

7.

39. 26. The composition of claim 25, wherein the encoded inhibitor of TGF-beta activity comprises a protein that binds to the TGF-beta receptor, and the protein that binds to the TGF-beta receptor comprises TGF-beta fusion 2 (TGFbf2) protein.

40. 40. The composition of claim 39, wherein the TGFbf2 comprises a human IgE signal peptide and TGF-beta variant 2 (TGFbv2).

41. 41. The composition of claim 40, wherein the human IgE signal peptide comprises an amino acid sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

37.

42. 41. The composition of claim 40, wherein the human IgE signal peptide comprises the amino acid sequence set forth in SEQ ID NO:

37.

43. 41. The composition of claim 40, wherein the TGFbv2 comprises an amino acid sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

8.

44. 41. The composition of claim 40, wherein the TGFbv2 comprises the sequence set forth in SEQ ID NO:

8.

45. 25. The composition of claim 24, wherein the sequence encoding the cytokine comprises a second promoter region that drives expression of the cytokine, and the sequence encoding the TGF-beta activity inhibitor comprises a third promoter region that drives expression of the TGF-beta activity inhibitor.

46. 46. ​​The composition of claim 45, wherein the second promoter region and the third promoter region each comprise a late promoter.

47. 47. The composition of claim 46, wherein the late promoter comprises any one of SSP, P7.5, P28, P135, TK promoter, E / L, F7L, H5R, H1L, A1L, J3R, E4L, I1L, I5L, I7L, T7, I2L, FP4b, ATI, P11, PFL1, PH5, L4R, 28kDa promoter, or any variation or combination thereof.

48. 47. The composition of claim 46, wherein the late promoter comprises a weak late promoter.

49. 49. The composition of claim 48, wherein the weak late promoter comprises a P135 promoter.

50. 47. The composition of claim 46, wherein the second promoter region comprises a P135 promoter.

51. 47. The composition of claim 46, wherein the late promoter comprises a strong late promoter.

52. 52. The composition of claim 51 , wherein the strong late promoter comprises a P7.5 promoter.

53. 47. The composition of claim 46, wherein the third promoter region comprises a P7.5 promoter.

54. 25. The composition of claim 24, wherein the exogenous nucleic acid encoding the cytokine or a functional variant thereof, the exogenous nucleic acid encoding the TGF-beta activity inhibitor, and the exogenous nucleic acid encoding the chemokine receptor or a functional variant thereof are located in a single genome.

55. 55. The composition of any one of claims 1 to 54, wherein the vector is an oncolytic virus, and the oncolytic virus comprises at least one genome modification.

56. 56. The composition of claim 55, wherein the at least one alteration comprises a mutation or deletion of at least one gene selected from the group consisting of thymidine kinase (TK) 56, F13L, A36R, A34R, A33R, A52R, B5R, B8R, B18R, SPI-1, SPI-2, B15R, VGF, E3L, K3L, A41L, K7R, or N1L, a functional fragment thereof, or any combination thereof.

57. 57. The composition of claim 56, wherein the at least one modification comprises a deletion of genes A52R and TK.

58. 56. The composition of claim 55, wherein the oncolytic virus is a poxvirus, adeno-associated virus, adenovirus, reovirus, lentivirus, herpes simplex virus, vesicular stomatitis virus, mengovirus, myxoma virus, Newcastle disease virus, measles virus, or poliovirus.

59. 59. The composition of claim 58, wherein the poxvirus is a vaccinia virus.

60. 60. The composition of claim 59, wherein the vaccinia virus is a Western Reserve strain.

61. 56. The composition of claim 55, wherein the at least one genomic modification results in about a 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 12-fold, about 14-fold, about 16-fold, about 18-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, about 55-fold, about 60-fold, about 65-fold, about 70-fold, about 75-fold, about 80-fold, about 85-fold, about 90-fold, about 95-fold, or about 100-fold increase in the efficacy of tumor-targeted systemic delivery of the virus.

62. a first polypeptide comprising interleukin-12 (IL-12) or a functional variant thereof; and A second polypeptide comprising an inhibitor of transforming growth factor beta (TGF-beta) activity. A nucleic acid comprising a sequence encoding

63. 63. The nucleic acid of claim 62, comprising DNA or RNA.

64. 63. The nucleic acid of claim 62, wherein the IL-12 is mouse IL-12 or human IL-12.

65. 63. The nucleic acid of claim 62, wherein the IL-12 comprises an alpha subunit and a beta subunit.

66. 66. The nucleic acid of claim 65, wherein the first polypeptide comprising the IL-12 alpha subunit and the IL-12 beta subunit further comprises a linker.

67. 66. The nucleic acid of claim 65, wherein the IL-12 alpha subunit comprises an amino acid sequence having at least 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NO:13 or SEQ ID NO:

16.

68. 66. The nucleic acid of claim 65, wherein the IL-12 alpha subunit comprises the sequence set forth in any one of SEQ ID NO: 13 or SEQ ID NO:

16.

69. 66. The nucleic acid of claim 65, wherein the IL-12 beta subunit comprises an amino acid sequence having at least 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NO:14 or SEQ ID NO:

17.

70. 66. The nucleic acid of claim 65, wherein the IL-12 beta subunit comprises the amino acid sequence set forth in any one of SEQ ID NO: 14 or SEQ ID NO:

17.

71. 67. The nucleic acid of claim 66, wherein the linker comprises an amino acid sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

18.

72. 67. The nucleic acid of claim 66, wherein the linker comprises the amino acid sequence set forth in SEQ ID NO:

18.

73. 63. The nucleic acid of claim 62, wherein the encoded IL-12 comprises an amino acid sequence having at least 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NO:12 or SEQ ID NO:

15.

74. 63. The nucleic acid of claim 62, wherein the encoded IL-12 comprises the amino acid sequence set forth in any one of SEQ ID NO: 12 or SEQ ID NO:

15.

75. 63. The nucleic acid of claim 62, wherein the sequence encoding IL-12 comprises a nucleic acid sequence having at least 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NO:64 or SEQ ID NO:

66.

76. 63. The nucleic acid of claim 62, wherein the sequence encoding IL-12 comprises the nucleic acid sequence set forth in any one of SEQ ID NO:64 or SEQ ID NO:

66.

77. 63. The nucleic acid of claim 62, wherein the inhibitor of TGF-beta activity comprises a TGF-beta dominant negative, a TGF-beta receptor dominant negative, a protein that binds to TGF-beta, or a protein that binds to a TGF-beta receptor.

78. 78. The nucleic acid of claim 77, wherein the inhibitor of TGF-beta activity comprises a protein that binds to TGF-beta, and the protein that binds to TGF-beta is an antibody or a functional variant thereof.

79. 78. The nucleic acid of claim 77, wherein the inhibitor of TGF-beta activity comprises a protein that binds to the TGF-beta receptor, and the protein that binds to the TGF-beta receptor is an antibody or a functional variant thereof.

80. The nucleic acid of claim 77, wherein the inhibitor of TGF-beta activity comprises a protein that binds to the TGF-beta receptor, and the protein that binds to the TGF-beta receptor is a protein that includes a TGF-beta domain.

81. 81. The nucleic acid of claim 80, wherein the protein comprising the TGF-beta domain comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 9.

82. 81. The nucleic acid of claim 80, wherein the protein comprising a domain of TGF-beta comprises an amino acid sequence having at least 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NO:7 or SEQ ID NO:

8.

83. 81. The nucleic acid of claim 80, wherein the protein comprising the domain of TGF-beta comprises the amino acid sequence set forth in any one of SEQ ID NO: 7 or SEQ ID NO:

8.

84. 63. The nucleic acid of claim 62, wherein the sequence encoding the inhibitor of transforming growth factor beta (TGF-beta) activity comprises a nucleic acid sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

67.

85. 63. The nucleic acid of claim 62, wherein the sequence encoding the inhibitor of TGF-beta activity comprises the nucleic acid sequence set forth in SEQ ID NO:

67.

86. 78. The nucleic acid of claim 77, comprising a protein that binds to the TGF-beta receptor, wherein the protein that binds to the TGF-beta receptor comprises TGF-beta fusion 1 (TGFbf1) protein.

87. 87. The nucleic acid of claim 86, wherein the TGFbf1 protein comprises a mouse IL-2 signal peptide and TGF-beta variant 1 (TGFbv1).

88. 88. The nucleic acid of claim 87, wherein the mouse IL-2 signal peptide comprises an amino acid sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

36.

89. 88. The nucleic acid of claim 87, wherein the mouse IL-2 signal peptide comprises the amino acid sequence set forth in SEQ ID NO:

36.

90. 88. The nucleic acid of claim 87, wherein the TGFbv1 comprises an amino acid sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

7.

91. 88. The nucleic acid of claim 87, wherein the TGFbv1 comprises the sequence set forth in SEQ ID NO:

7.

92. 78. The nucleic acid of claim 77, comprising a protein that binds to the TGF-beta receptor, wherein the protein that binds to the TGF-beta receptor comprises the TGF-beta fusion 2 (TGFbf2) protein.

93. 93. The nucleic acid of claim 92, wherein the TGFbf2 comprises a human IgE signal peptide and TGF-beta variant 2 (TGFbv2).

94. 94. The nucleic acid of claim 93, wherein the human IgE signal peptide comprises an amino acid sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

37.

95. 94. The nucleic acid of claim 93, wherein the human IgE signal peptide comprises the amino acid sequence set forth in SEQ ID NO:

37.

96. 94. The nucleic acid of claim 93, wherein the TGFbv2 comprises an amino acid sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

8.

97. 94. The nucleic acid of claim 93, wherein the TGFbv2 comprises the sequence set forth in SEQ ID NO:

8.

98. 78. The nucleic acid of claim 77, wherein the protein that binds to the TGF-beta receptor binds to TGF-beta receptor II but not to TGF-beta receptor I.

99. 63. The nucleic acid of claim 62, further comprising at least one promoter region.

100. 100. The nucleic acid of claim 99, wherein said at least one promoter region drives expression of at least two of said polypeptides.

101. 100. The nucleic acid of claim 99, wherein the at least one promoter region comprises a first promoter region and a second promoter region, the first promoter region driving expression of the polypeptide comprising the IL-12, and the second promoter region driving expression of the TGF-beta activity inhibitor.

102. 102. The nucleic acid of claim 101, wherein the first promoter region and the second promoter region each comprise any one of SSP, P7.5, P28, P135, TK promoter, E / L, F7L, H5R, H1L, A1L, J3R, E4L, I1L, I5L, I7L, T7, I2L, FP4b, ATI, P11, PFL1, PH5, L4R, 28 kDa promoter, or any variation or combination thereof.

103. 103. The nucleic acid of claim 102, wherein the first promoter region comprises a P7.5 promoter.

104. 103. The nucleic acid of claim 102, wherein the first promoter region comprises a P135 promoter.

105. 103. The nucleic acid of claim 102, wherein the second promoter region comprises a P28 promoter.

106. 103. The nucleic acid of claim 102, wherein the second promoter region comprises a P7.5 promoter.

107. 107. The nucleic acid of any one of claims 62 to 106, further comprising a sequence encoding a third polypeptide comprising a chemokine receptor or a functional variant thereof.

108. 108. The nucleic acid of claim 107, wherein the chemokine receptor comprises at least one of a CXC receptor, a CC receptor, a CX3C receptor, an XC receptor, a functional fragment thereof, a functional variant thereof, or any combination thereof.

109. 108. The nucleic acid of claim 107, wherein the chemokine receptor comprises at least one of CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, CX3CR1, XCR1, a functional fragment thereof, or a functional variant thereof, or any combination thereof.

110. The nucleic acid of claim 109, wherein the chemokine receptor comprises CXCR3.

111. 111. The nucleic acid of claim 110, wherein the chemokine receptor comprises an amino acid sequence having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:42 or SEQ ID NO:

43.

112. The nucleic acid of claim 110, wherein the chemokine receptor comprises the amino acid sequence set forth in SEQ ID NO: 42 or SEQ ID NO:

43.

113. 108. The nucleic acid of claim 107, wherein said third polypeptide comprises a third promoter region that causes expression of said chemokine receptor prior to expression of said IL-12 and said inhibitor of TGF-beta activity.

114. 114. The nucleic acid of claim 113, wherein the third promoter region comprises any one of A52R, pB8, mH5, I4L, LEO, pF11, I3L, P7.5, TK promoter, F7L, H5R, a short synthetic promoter (SSP), or any variation or combination thereof.

115. 115. The nucleic acid of claim 114, wherein the third promoter comprises an A52R promoter.

116. a first region encoding a first polypeptide comprising an amino acid sequence having at least 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NO:12 or SEQ ID NO:15; a second region encoding a second polypeptide comprising an amino acid sequence having at least 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NO:40 or SEQ ID NO:41; A nucleic acid comprising:

117. 117. The nucleic acid of claim 116, comprising DNA or RNA.

118. 117. The nucleic acid of claim 116, further comprising at least one promoter region.

119. 120. The nucleic acid of claim 119, wherein said at least one promoter region drives expression of at least two of said polypeptides.

120. 120. The nucleic acid of claim 119, comprising a first promoter region and a second promoter region, wherein the first promoter region drives expression of the first polypeptide and the second promoter region drives expression of the second polypeptide.

121. 121. The nucleic acid of claim 120, wherein the first promoter region and the second promoter region each comprise any one of SSP, P7.5, P28, P135, TK promoter, E / L, F7L, H5R, H1L, A1L, J3R, E4L, I1L, I5L, I7L, T7, I2L, FP4b, ATI, P11, PFL1, PH5, L4R, 28 kDa promoter, or any variation or combination thereof.

122. 121. The nucleic acid of claim 120, wherein the first promoter region comprises a P7.5 promoter.

123. 121. The nucleic acid of claim 120, wherein the first promoter region comprises a P135 promoter.

124. 121. The nucleic acid of claim 120, wherein the second promoter region comprises a P28 promoter.

125. 121. The nucleic acid of claim 120, wherein the second promoter region comprises a P7.5 promoter.

126. the first polypeptide comprises the sequence set forth in any one of SEQ ID NO: 12 or SEQ ID NO: 15; 117. The nucleic acid of claim 116, wherein the second polypeptide comprises the sequence set forth in any one of SEQ ID NO: 40 or SEQ ID NO:

41.

127. the first region has a nucleic acid sequence comprising at least 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NO:64 or SEQ ID NO:66; 117. The nucleic acid of claim 116, wherein the second region has a nucleic acid sequence comprising at least 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NO:65 or SEQ ID NO:

67.

128. The nucleic acid comprises DNA, and the DNA comprises, in 5' to 3' order: a first region encoding IL-12 or a functional variant thereof; and A second region encoding an inhibitor of TGF-beta activity 118. The nucleic acid of claim 117, comprising a sequence comprising:

129. The nucleic acid of claim 117, wherein the nucleic acid comprises DNA, and the DNA comprises, in 5' to 3' order, a sequence encoding the amino acid sequences set forth in SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 36, and SEQ ID NO:

7.

130. The nucleic acid of claim 117, wherein the nucleic acid comprises DNA, and the DNA comprises, in 5' to 3' order, a sequence encoding the amino acid sequences set forth in SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 16, SEQ ID NO: 37, and SEQ ID NO:

8.

131. The nucleic acid of claim 117, wherein the nucleic acid comprises DNA, and the DNA comprises, in 5' to 3' order, a sequence encoding the amino acid sequences set forth in SEQ ID NO: 12 and SEQ ID NO:

40.

132. The nucleic acid of claim 117, wherein the nucleic acid comprises DNA, and the DNA comprises, in 5' to 3' order, a sequence encoding the amino acid sequences set forth in SEQ ID NO: 15 and SEQ ID NO:

41.

133. 133. The nucleic acid of any one of claims 116-132, wherein the nucleic acid further comprises a third region encoding a third polypeptide comprising an amino acid sequence having at least 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NO:42 or SEQ ID NO:

43.

134. 134. The nucleic acid of claim 133, wherein the third polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NO: 42 or SEQ ID NO:

43.

135. 133. The nucleic acid of any one of claims 116 to 132, further comprising a third region comprising a nucleic acid sequence having at least 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NO:69 or SEQ ID NO:

86.

136. 136. The nucleic acid of claim 135, wherein the third region comprises a nucleic acid sequence set forth in any one of SEQ ID NO: 69 or SEQ ID NO:

86.

137. 134. The nucleic acid of Claim 133, wherein the third region encoding the third polypeptide further comprises a third promoter region that effects expression of the third polypeptide prior to expression of the first polypeptide and the second polypeptide.

138. 138. The nucleic acid of claim 137, wherein the third promoter comprises any one of A52R, pB8, mH5, I4L, LEO, pF11, I3L, P7.5, TK promoter, F7L, H5R, a short synthetic promoter (SSP), or any variation or combination thereof.

139. 139. The nucleic acid of claim 138, wherein the third promoter comprises an A52R promoter.

140. 140. The nucleic acid of any one of claims 116 to 139, which is present in an oncolytic virus.

141. 141. The nucleic acid of claim 140, wherein the oncolytic virus is a poxvirus, adeno-associated virus, adenovirus, reovirus, lentivirus, herpes simplex virus, vesicular stomatitis virus, mengovirus, myxoma virus, Newcastle disease virus, measles virus, or poliovirus.

142. 142. The nucleic acid of claim 141, wherein the poxvirus is a vaccinia virus.

143. 143. The nucleic acid of claim 142, wherein the vaccinia virus is Western Reserve vaccinia virus (ATCC VR-1354), vaccinia virus Ankara (ATCC VR-1508), vaccinia virus Ankara (ATCC VR-1566), vaccinia virus strain Wyeth (ATCC VR-1536), or a modified strain of vaccinia virus Wyeth (ATCC VR-325).

144. 141. The nucleic acid of claim 140, which is inserted into a viral genome.

145. 141. The nucleic acid of claim 140, further comprising a mutation or deletion in at least one viral gene selected from the group consisting of thymidine kinase (TK), F13L, A36R, A34R, A33R, A52R, B5R, B8R, B18R, SPI-1, SPI-2, B15R, VGF, E3L, K3L, A41L, K7R, or N1L, a functional fragment thereof, or any combination thereof.

146. In the order 5' to 3', a first promoter region, wherein the promoter comprises an A52R promoter; Insertion of the first region encoding human CXCR3 An insertion at the A52R locus comprising In the order 5' to 3', a second promoter region, wherein the promoter comprises P135; a second region encoding human IL-12, a third promoter region, the promoter of which comprises P7.5; and A third region encoding a TGF-beta variant An insertion at the TK locus comprising A nucleic acid molecule comprising:

147. The nucleic acid molecule of claim 146, wherein the P135 promoter comprises the nucleic acid sequence set forth in SEQ ID NO: 56 and the P7.5 promoter comprises the nucleic acid sequence set forth in SEQ ID NO:

57.

148. the first region encoding human CXCR3 comprises the nucleic acid sequence set forth in SEQ ID NO: 86; the second region encoding human IL-12 comprises the nucleic acid sequence set forth in SEQ ID NO: 66; The nucleic acid of claim 146, wherein the third region encoding a TGF-beta inhibitor comprises the nucleic acid sequence set forth in SEQ ID NO:

67.

149. an insertion in the A52R locus comprising the sequence set forth in SEQ ID NO: 88; an insertion in the TK locus comprising the sequence set forth in SEQ ID NO: 85; A nucleic acid molecule comprising:

150. A composition according to any one of claims 1 to 61 or a nucleic acid according to any one of claims 62 to 149, a pharmaceutically acceptable excipient; 10. A pharmaceutical composition comprising:

151. 151. The pharmaceutical composition of claim 150, in liquid dosage form.

152. 151. The pharmaceutical composition of claim 150, wherein the pharmaceutically acceptable excipient is buffered saline.

153. 153. The pharmaceutical composition of claim 152, wherein the buffered saline is phosphate buffered saline (PBS), Dulbecco's PBS (DPBS), TRIS buffered saline (TBS), Hank's balanced salt solution (HBSS), Earl's balanced salt solution (EBSS), standard citrate saline (SSC), HEPES buffered saline (HBS), or Gey's balanced salt solution.

154. 151. The pharmaceutical composition of claim 150, wherein the composition further comprises a liposome or nanoparticle.

155. 155. The pharmaceutical composition of claim 154, wherein the nucleic acid or vector is associated with the liposome or nanoparticle.

156. 156. A method for the treatment of cancer, comprising the step of administering to a subject having cancer the pharmaceutical composition of any one of claims 150 to 155 in an amount sufficient to treat the cancer.

157. 157. The method of claim 156, wherein the cancer is a solid tumor, leukemia, or lymphoma.

158. 157. The method of claim 156, wherein the cancer comprises melanoma, hepatocellular carcinoma, breast cancer, lung cancer, peritoneal cancer, prostate cancer, bladder cancer, ovarian cancer, leukemia, lymphoma, kidney cancer, pancreatic cancer, epithelial carcinoma, gastric cancer, colon cancer, duodenal cancer, pancreatic adenocarcinoma, mesothelioma, glioblastoma multiforme, astrocytoma, multiple myeloma, prostate cancer, hepatocellular carcinoma, bile duct sarcoma, pancreatic adenocarcinoma, head and neck squamous cell carcinoma, colorectal cancer, gastrointestinal-type gastric adenocarcinoma, cervical squamous cell carcinoma, osteosarcoma, epithelial ovarian cancer, acute lymphoblastic lymphoma, myeloproliferative neoplasm, or sarcoma.

159. 157. The method of claim 156, wherein the administering step comprises intratumoral administration.

160. 157. The method of claim 156, wherein the administering step comprises systemic administration.

161. 161. The method of claim 160, wherein the systemic administration comprises oral administration, parenteral administration, intranasal administration, sublingual administration, rectal administration, transdermal administration, or any combination thereof.

162. 156. A method for activating an anti-cancer immune response, comprising administering to a subject with cancer the pharmaceutical composition of any one of claims 150 to 155.

163. 163. The method of claim 162, wherein the cancer is a solid tumor, leukemia, or lymphoma.

164. 163. The method of claim 162, wherein the cancer comprises melanoma, hepatocellular carcinoma, breast cancer, lung cancer, peritoneal cancer, prostate cancer, bladder cancer, ovarian cancer, leukemia, lymphoma, kidney cancer, pancreatic cancer, epithelial carcinoma, gastric cancer, colon cancer, duodenal cancer, pancreatic adenocarcinoma, mesothelioma, glioblastoma multiforme, astrocytoma, multiple myeloma, prostate cancer, hepatocellular carcinoma, bile duct sarcoma, pancreatic adenocarcinoma, head and neck squamous cell carcinoma, colorectal cancer, gastrointestinal-type gastric adenocarcinoma, cervical squamous cell carcinoma, osteosarcoma, epithelial ovarian cancer, acute lymphoblastic lymphoma, myeloproliferative neoplasm, or sarcoma.

165. 163. The method of claim 162, wherein the administering step is intratumoral administration.

166. 163. The method of claim 162, wherein the administering step is systemic administration.

167. 167. The method of claim 166, wherein the systemic administration comprises oral administration, parenteral administration, intranasal administration, sublingual administration, rectal administration, transdermal administration, or any combination thereof.

168. 163. The method of claim 162, wherein the anti-cancer response is defined by increased infiltration of CD3+ CD8+ T cells into the tumor.

169. 169. The method of claim 168, wherein the increased infiltration of CD3+ CD8+ T cells is about a 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold increase in CD3+ CD8+ T cells in the tumor compared to an untreated tumor.

170. 169. The method of claim 168, wherein the increased infiltration of CD3+ CD8+ T cells in the tumor is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% greater than in an untreated tumor.

171. 163. The method of claim 162, wherein the anti-tumor immune response comprises increased expression of one or more genes associated with interferon-gamma (IFNG).

172. 172. The method of claim 171, wherein the one or more genes associated with IFNG are selected from the group consisting of CXCL11, XCR1, STAT1, IDO1, IL12B, IFNG, CIITA, H2-EB1, H2-AB1, TBX21, CXCR3, CD2, LTB, CXCL16, B2M, VCAM1, TAP1, IFIT2, TAP2, IL2RG, STAT2, CD274, and IRF1.

173. The method of claim 171, wherein the increase in expression of one or more genes associated with IFNG is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 120%, about 140%, about 160%, about 180%, about 200%, about 250%, about 300%, about 350%, or about 400% compared to the median expression across treated and untreated cells.

174. 163. The method of claim 162, wherein the anti-cancer immune response comprises a decrease in the expression of one or more genes associated with TGFB1.

175. The method of claim 174, wherein the one or more genes associated with TGFB1 are selected from the group consisting of ILz1B, LPL, SLP1, FBN1, LCN2, CXCL5, OGN, PLOD2, TNFAIP6, CAN, ABCG1, ACKR3, and COL15A1.

176. 175. The method of claim 174, wherein the decrease in expression of one or more genes associated with TGFB1 is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to the median expression across treated and untreated cells.

177. 163. The method of claim 162, wherein the anti-cancer response comprises an increase in granzyme B expression.

178. The method of claim 177, wherein the increase in Granzyme B expression is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 125%, about 150%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000% greater than in untreated cells.

179. 1. A method for reducing the incidence of tumor cell growth, comprising: administering to the tumor cells a pharmaceutical composition according to any one of claims 150 to 155 in an effective amount sufficient to reduce the incidence of tumor cell growth. A method comprising:

180. 180. The method of claim 179, wherein the tumor cells are derived from a solid tumor, leukemia, or lymphoma.

181. 180. The method of claim 179, wherein the tumor cells are derived from melanoma, hepatocellular carcinoma, breast cancer, lung cancer, peritoneal cancer, prostate cancer, bladder cancer, ovarian cancer, leukemia, lymphoma, kidney cancer, pancreatic cancer, epithelial carcinoma, gastric cancer, colon cancer, duodenal cancer, pancreatic adenocarcinoma, mesothelioma, glioblastoma multiforme, astrocytoma, multiple myeloma, prostate cancer, hepatocellular carcinoma, bile duct sarcoma, pancreatic adenocarcinoma, head and neck squamous cell carcinoma, colorectal cancer, gastrointestinal-type gastric adenocarcinoma, cervical squamous cell carcinoma, osteosarcoma, epithelial ovarian cancer, acute lymphoblastic lymphoma, myeloproliferative neoplasm, or sarcoma.

182. 180. The method of claim 179, wherein the administering step is intratumoral administration.

183. 180. The method of claim 179, wherein the administering step is systemic administration.

184. 184. The method of claim 183, wherein the systemic administration comprises oral administration, parenteral administration, intranasal administration, sublingual administration, rectal administration, transdermal administration, or any combination thereof.

185. 180. The method of claim 179, wherein the incidence of tumor cell growth is reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or about 100% compared to the incidence of untreated tumor cell growth.

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